Neuroblastoma (NB) is the most common extracranial solid tumor diagnosed in the first year of life. The disease is characterized by lack of somatic mutations and high genomic instability including the amplification of MYCN which correlates with poor clinical outcome. These features of NB suggest a pivotal role of transcriptome dynamics in this disease. Analyses of 1089 NB patients show that an overall increase in m6A-regulatory factors correlates with the worst prognosis. Notably, the m6A reader YTHDF1 is upregulated in stage 4 NB corresponding to the highest degree of MYCN amplification. In line with these notions, we observed a positive correlation between MYCN and YTHDF1 by analyzing 33 NB cell lines with different MYCN amplification status. Consistent with this observation, we demonstrated that MYCN knockdown is accompanied by a decrease in all the YTHDF proteins. Furthermore, we demonstrate a loss of proliferating activity in a MYCN-amplified cell line upon knocking out all the YTHDF paralogs and show that the loss in proliferation is mediated by downregulation of DNA replication and chromatin remodeling upon YTHDF depletion. Retinoic acid (RA) is a key agent employed in NB therapy; it induces cell differentiation limiting the proliferation of malignant cells. However, therapeutic protocols adopting RA are limited to maintenance therapy. Thereby, identifying new therapeutic strategies that synergistically enhance RA response is essential to expand the therapeutic window for this drug. Since m6A is a well-known regulator of cellular differentiation, we hypothesized that the protein involved in m6A regulation could act in concert with RA in driving this process. We demonstrate that YTHDF depletion potentiates the response to RA enriching the key processes required for differentiation and that this correlates with an enhanced pro-neural of the YTHDF depleted cells. Together, our findings suggest that targeting the YTHDF family in MYCN-amplified NB represents a promising therapeutic opportunity to control the progression of this disease.

Ime4 is the catalytic subunit of a conserved methyltransferase (MTase) complex found in yeast, S. cerevisiae. This complex is responsible for creating the RNA modification N6- methyladenosine (m6A), the most common post-transcriptional modification in higher eukaryotes. There is evidence to suggest that m6A is an important mediator of gene expression control within the cell and has been associated with a diverse array of phenotypic effects, notably as a conserved determinant of cell fate. The MTase complex is known to be a nuclear protein, the compartment where it is believed to carry out most of its methylation activity. Recently, the nuclear localization signals (NLS) of the subunits of the human MTase complex were experimentally identified, whereas the NLSs of the yeast MTase complex remain unknown. Here, we have experimentally identified the amino acid sequence 517RKYQEFMKSKTGTSHTGTKKIDKK540, located within the C-terminal region, as a putative bipartite NLS for Ime4.

Experiments were performed to test sequence and structural specific interactions of proteins with a conserved RNA modification enzyme, which is known as Ime4 in yeast and Mettl3 in mammals. Ime4 methylates N6-adenosine bases on mRNA molecules. The goal of this project is to gain direct insights into how novel proteins interact with Ime4 to form the methyltranferase (MTase) complex and to identify proteins that are essential for Ime4 activity. It has been recognized that there are two proteins that interact within the Ime4 complex, which are known as Mum2 (a cytoplasmic protein essential for meiotic DNA replication within yeast) and Slz1 (a transcription factor). We hypothesize that the N-terminal domain of Ime4 is the location of binding of the aforementioned proteins in this complex. Similarly, we tested whether the human ortholog of Ime4 (Mettl3) forms an analogous complex that includes an ortholog of Mum2, known as WTAP, and its binding partner WT1. The major approaches include in vivo genetic assays in yeast to test protein-protein interactions and the use of recombinant DNA technology to construct fusion genes/deletions. The results demonstrate that Mum2 interacts with a specific, non-conserved region in the Ime4 N-terminal domain. Furthermore, we discovered a new binding partner, Ygl036w, which also interacts with Ime4. Currently, several experiments are being carried out with the Mettl3 complex and its hypothesized protein binding partners to assess the interactions of this complex.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24756号 / 生博第497号 / 新制||生||66(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 北島 智也, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

The reward system is a network of structures in the brain responsible for the feelings of pleasure, motivation, and decision making. It is comprised of the prefrontal cortex, orbitofrontal cortex (OFC), nucleus accumbens, ventral tegmental area, amygdala and the hippocampus, brain regions that come together to process rewarding stimuli, commonly referred to as rewards, to positively shape behavior. Rewards are well known to induce a range of molecular changes within the reward system that mediate reinforcing effects of rewards—neuroadaptations. These neuroadaptations can not only support adaptive behavior but also can mediate negative symptoms of psychiatric disorders such as anhedonia, withdrawal, or drug tolerance. Hence, aberrant functioning of the reward circuitry is present in patients with psychiatric disorders such as addiction, bipolar disorder, eating disorders, major depressive disorder, and schizophrenia. The molecular mechanisms underlying the function of the reward system are not fully understood and therefore elucidating the reward-induced neuroadaptations could inform future therapeutic approaches for symptoms caused by aberrant reward processing associated with psychiatric disorders. This thesis aims to characterize two types of neuroadaptations, circular RNA (circRNA) transcriptomic changes as well as N6-methyladenosine (m6A) epitranscriptomic adaptations, in the context of appetitive reward and opioids, respectively. First, we focused on describing circRNA related neuroadaptations within the OFC, and their functional implications, in the context of sucrose seeking behavior. We reported the first circRNA profile associated with appetitive reward and identified a regulation of 92 OFC circRNAs by sucrose self-administration. Among these changes we observed a downregulation of circNrxn3, a circRNA originating from neurexin 3 (Nrxn3), a gene involved in synaptogenesis, learning, and memory. Transcriptomic profiling via RNA sequencing and qPCR of the OFC following in vivo knock-down of circNrxn3 revealed differential regulation of genes associated with pathways important for learning and memory and altered splicing of Nrxn3. Furthermore, circNrxn3 knock-down enhanced sucrose self-administration and motivation for sucrose. Using RNA-immunoprecipitation, we reported binding of circNrxn3 to the known Nrxn3 splicing factor SAM68. circNrxn3 is the first reported circRNA capable of regulating reward behavior. In addition, circNrxn3-mediated interactions with SAM68 may impact subsequent downstream processing of RNAs such as the regulation of gene expression and splicing. We then went to characterize m6A epitranscriptomic adaptations induced by a commonly misused drug, the opioid morphine. m6A modifications have not been studied in opioid use disorder, despite being the most common RNA modification. We detected significant regulation of m6A-modifying enzymes in rat primary cortical cultures following morphine treatment, including AlkB Homolog 5 (Alkbh5). The m6a demethylase Alkbh5 functions as an m6A eraser, removing m6A modifications from mRNA. We hypothesized that chronic opioid exposure regulates m6A modifications through modulation of Alkbh5 and profiled m6A modifications in primary cortical cultures following chronic morphine exposure and Alkbh5 knock-down. We observed differential regulation of m6A modifications for 568 transcripts following morphine and 2865 following Alkbh5 knock-down. 103 transcripts were commonly regulated by both morphine and Alkbh5 knock-down, and the two treatments elicited concordant m6A epitranscriptomic profiles, suggesting that a subset of morphine-driven m6A modifications may be mediated through downregulation of Alkbh5 in cortical cultures. Together, this volume expands our understanding of molecular neuroadaptations induced by both appetitive reward and opioids. We have identified potential facilitators that could impact reward seeking, motivation and drug induced molecular adaptations that could inform future studies. / Biology

The N6-methyladenosine, also known as m6A, is the most common post-transcriptional modification in mRNAs and long non-coding RNAs and that profoundly influences mRNA biology, from early processing in the nucleus to final steps of translation and decay in the cytoplasm. Taking into consideration the importance of RNA in shaping cell fate, m6A is widely recognized as an additional layer in the regulation of gene expression, also thanks to its dynamic and reversible nature. Therefore, it is not surprising that any misregulation in m6A content might lead to the loss of cellular homeostasis. This effect is particularly evident when it comes to stem cells differentiation, embryo development and cancer. In a tumorigenic context, the m6A could affect the development, progression, cancer stem cells (CSCs) renewal and drug resistance of solid and liquid tumours. So, the m6A is consistently becoming a new attractive pharmacological target. Neuroblastoma (NB) is a neuroendocrine tumour of early childhood that derives from undifferentiated cells of the sympathoadrenal lineage of the neural crest. About 50% of patients have a very aggressive form of NB, with an overall survival rate of less than 30% despite heavy treatments. Moreover, NB is a challenging druggable tumour due to a low rate of somatic mutations. Somatic mutations at significant frequency have been identified in only five genes that also show detectable expression. Among these, only one is currently a directly validated druggable target. Two m6A regulators (METTL14 and ALKBH5) are aberrantly expressed in high-risk NB patients, and their alteration in NB cell lines affects tumour aggressiveness. Specifically, the overexpression of the methyltransferase METTL14 increases cell proliferation and invasion in vitro and tumour growth in mice acting as an oncogene, while ALKBH5 restoration affects cell proliferation, apoptosis and invasion in an opposite fashion. Importantly, the demethylase ALKBH5 impaired tumour formation in vivo when costitutively expressed and dramatically slows down tumor progression in mice when is induced by causing massive apoptosis. These data suggest that ALKBH5 acts as a potent tumour suppressor in NB. We discovered that METTL14 and ALKBH5 exert their effect on different levels by affecting mRNA stability or translation, respectively. Although the contribution to NB of the altered stability of transcripts related to mRNA processing in METTL14-overexpressing cells is less understand, the increase translation of pro-apoptotic genes in the ALKBH5-overexpression condition leaves little doubts. Our results unveil the m6A and its regulators as potential therapeutic targets for treating NB. Indeed, in collaboration with the Laboratory of Genomic Screening of Professor Alessandro Provenzani, we presented an encouraging proof-of-concept of the reader YTHDF1 as a possible pharmacological target.

No description available.

In the yeast Saccharomyces cerevisiae, the IME4 methyltransferase, interacts genetically with methyl binding protein, Pho92, to affect the expression of PHO regulon target genes. Cells mutant in IME4 or PHO92 show increases in the RNA abundance of PHO regulon target genes. The increase in the RNA abundance of the PHO regulon target genes is not additive in the cells double mutant in IME4 and PHO92. Hence, Ime4 and Pho92 interact in a single pathway in PHO regulon. Surprisingly, cells overexpressing IME4 and MUM2 shows increase in some PHO regulon target genes, indicating that IME4 affects the PHO regulon target genes through multiple mechanisms in different conditions. A promoter swap experiment revealed that one of the PHO regulon mRNAs that codes for phosphatase, PHO5, is a direct target of Ime4. Further experiments are required to examine whether the same is true for all PHO regulon mRNAs.

Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22046号 / 薬科博第112号 / 新制||薬科||12(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 准教授 Fustin,Jean Michel, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM