This is the first of 2 review papers on genetics and genomics appearing as part of the series on “omics.” Genomics pertains to all components of an organism’s genes, whereas genetics involves analysis of a specific gene(s) in the context of heredity. The paper provides introductory comments, describes the basis of human genetic diversity, and addresses the phenotypic consequences of genetic variants. Rare variants with large effect sizes are responsible for single gene disorders, whereas complex polygenic diseases are typically due to multiple genetic variants, each exerting a modest effect size. To illustrate the clinical implications of genetic variants with large effect sizes, 3 common forms of hereditary cardiomyopathies are discussed as prototypic examples of single-gene disorders, including their genetics, clinical manifestations, pathogenesis, and treatment. The genetic basis of complex traits is discussed in a separate paper.

Long ncRNAs (non-protein coding transcripts generally considered longer than 200 nucleotides to be distinguished from classes of small RNAs) are abundantly transcribed from the mammalian genome. Despite their abundance, little is known about these transcripts. Although several individual long ncRNAs have been well-characterised and ascribed important cellular functions, there remains considerable controversy as to whether long ncRNAs are, in the main, functional. Indeed, their abundance has prompted many people to argue that long ncRNAs are simply transcriptional ‘noise’ generated by spurious transcription initiation events resulting from low RNA polymerase II fidelity. This thesis demonstrates that large numbers of long ncRNAs are specifically expressed along both temporal and spatial axes of mouse development in a manner consistent with a biological function. Custom-designed microarrays were employed to analyse the expression profiles of large numbers of long ncRNAs, along with protein-coding genes, in two models of cellular differentiation; the differentiation of mouse embryonic stem (ES) cells from pluripotency to differentiation along a hemopoietic lineage; and the commitment and differentiation of neural stem cells to oligodendrocytes. The core networks that include gene expression, transcription factor binding sites and chromatin domains that regulate ES cell pluripotency and lineage specification have been the subject of considerable attention and provide a detailed context in which to analyse ncRNA expression. Of those ncRNAs examined, 945 (26% of total) ncRNAs were expressed during the differentiation of ES to embryoid body (EB), of which 174 were significantly differentially expressed. Many of these ncRNAs were transcribed from genomic locations that overlapped modified chromatin domains, and in two further studied cases directly engaged with epigenetic machinery. Similarly, 332 long ncRNAs (9% of those examined) were expressed during processes of neuronal-glial fate switching, neurogenesis and oligodendrocyte progressive differentiation and termination, of which around half were also significantly differentially expressed. Furthermore, many of these ncRNAs exhibited expression profiles that coincided with pivotal events during the commitment and differentiation of neural stem cells (NSC) to mature myelinating oligodendrocytes. Consideration of the genomic context revealed many long ncRNAs were expressed from diverse places including intergenic, intronic, and imprinted loci and may overlap with, or are transcribed antisense to, protein-coding genes with previously described roles in either ES or NSC pluripotency and differentiation. This association also extended to expression profiles, where a comparative analysis often showed complex relationships of expression between ncRNAs and associated protein coding genes, suggesting a potential role for ncRNAs in regulating the expression of associated gene loci. The complexity and specificity of the long ncRNAs expression was illustrated by analysis of the in situ hybridisation (ISH) data conducted in collaboration with the Allen Brain Atlas. Of 1328 long ncRNAs, 849 (64%) were expressed in the mouse brain, 623 (47%) of which exhibited specific expression profiles associated with distinct neuroanatomical regions, cell types, or subcellular compartments. Again, examination of their genomic context revealed long ncRNAs were often associated with protein-coding genes of neurological importance and this association often extended to include linked expression profiles in the mouse brain. The comparative analysis of protein-coding gene expression relative to associated noncoding transcription also revealed an additional level of complexity in gene structure and genomic architecture. Analysis of both microarray and ISH data show 3’UTRs can exhibit discordant expression profiles relative to their associated protein coding genes, often in a tissue- and developmentally-specific manner. Indeed, a genome-wide analysis showed that the independent expression of 3’UTR transcripts is prevalent throughout the mouse genome where they may function intrinsically as long ncRNAs during development. Together, these genome-wide analyses indicate a large proportion of long ncRNAs exhibit specific expression profiles that are inconsistent with the notion they are meaningless transcriptional noise. Taken together with numerous studies published in recent years, this thesis provides evidence to support the emergence of long ncRNAs as a major functional component of the regulatory network that underpins differentiation and development in mammals and other complex organisms.

The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Though it is primarily known as the site of ribosomal biogenesis, the nucleolus is also capable of orchestrating the immobilization of a broad range of proteins under specific environmental conditions. This process, known as nucleolar sequestration, contributes to cell viability under stress. Despite the importance of this post-translational regulatory pathway, very little is known about the mechanisms that govern it. Here, we show that heat shock and acidosis, two physiological stimuli associated with nucleolar sequestration, induce the expression of long noncoding RNA (lncRNA) from stimulus-specific loci of the ribosomal intergenic spacer (IGS). These lncRNAs, in turn, immobilize proteins encoding a nucleolar detention sequence (NoDS) within a compartment of the nucleolus termed the detention centre (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate (ANS)-positive hydrophobic signature. Its formation is accompanied by a redistribution of nucleolar factors and an arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA, by environmental signals, operates as a molecular switch that regulates the structure and function of the nucleolus.

Long noncoding RNAs (lncRNAs) are being realized as important players in gene regulation and their misregulation has been considered as one of the underlying causes for tumor initiation and progression in many human pathologies. In the current thesis, I have addressed the functional role of lncRNAs in development and disease model systems. Genomic imprinting is an epigenetic phenomenon by which subset of genes are expressed in a parent of origin-specific manner. The Kcnq1 imprinted locus is epigenetically regulated by Kcnq1ot1 lncRNA. Deletion of an 890bp region at the 5’ end of Kcnq1ot1 in mouse resulted in the loss of silencing of neighboring ubiqui-tously imprinted genes (UIGs). In addition, we observed loss of DNA methylation at the UIG promoters. We have shown that Kcnq1ot1 RNA establishes CpG methylation by interacting with DNMT1. To explore the stability of lncRNA mediated silencing pathways, we have conditionally deleted Kcnq1ot1 in the mouse in a stage and tissue-specific manner. We have shown that Kcnq1ot1 is continuously required for maintaining the silencing of UIGs, whereas the silencing of the placental im-printed genes is maintained in an RNA independent manner.   To identify chromatin-associated lncRNA (CARs) on a genome-wide scale, we purified RNA from the sucrose gradient fractionated chromatin and subjected it to RNA sequencing. Our study has identified 141 intronic and 74 long intergenic CARs. Characterization of one of the CARs revealed that it regulates the expression of neighboring genes in cis by modulating the chromatin structure.   We have explored the functional role of lncRNA in tumor progression and initiation by using pediatric neuroblastoma. By transcriptional profiling of low- and high-risk tumors, we have identified several lncRNAs differentially expressed between these subtypes. We report an uncharacterized RNA NBAT-1, expressed at lower levels in high-risk tumors relative to low-risk tumors.  Using neuroblastoma cell culture system, we demonstrated that NBAT-1 has anti-cell proliferative and anti-invasive properties. In addition, it promotes differentiation of neurons from undifferentiated neuroblastoma cell lines.   In summary, by employing mouse genetics, cell culture based model system and expression profiling in tumors, we have uncovered new roles of lncRNA in gene regulation.

High throughput transcriptomic analyses have realized us with the fact that eukaryotic genome encodes thousands of noncoding RNAs (ncRNAs) with unknown function. In my thesis, I sought to address epigenetic regulation of transcription by ncRNA using the Kcnq1 imprinted cluster as a model system. Genomic imprinting is an epigenetic phenomenon whereby one of the parental alleles is silenced by epigenetic mechanism in a parent of origin-specific manner. A long ncRNA Kcnq1ot1 regulates imprinting of nearly 8 protein coding genes in the Kcnq1 imprinted cluster. Expression of Kcnq1ot1 is restricted to the paternal chromosome while that of protein-coding genes to the maternal chromosome. Kcnq1ot1 is a 91kb long, moderately stable, nuclear localized and RNAPII encoded transcript. We demonstrated that Kcnq1ot1 RNA itself mediates lineage specific silencing on the paternal chromosome by interacting with chromatin and recruiting the repressive chromatin modifiers to the imprinted gene promoters. Previously we identified an 890bp silencing domain (SD) at the 5´end of the Kcnq1ot1 RNA which is responsible for gene silencing. Targeted deletion of the 890SD in mouse resulted in specific loss of silencing of ubiquitously imprinted genes. We have further shown that Kcnq1ot1 interacts with Dnmt1 and recruit Dnmt1 at the somatic DMRs flanking some of the ubiquitously imprinted genes. We next addressed the stability of the Kcnq1ot1 mediated epigenetic silencing using transgenic mouse where we have conditionally deleted the Kcnq1ot1 RNA at different developmental stages and we found that Kcnq1ot1 RNA is required to maintain the silencing of the ubiquitously imprinted genes. In addition, DNA methylation, which controls imprinting of the ubiquitous genes require Kcnq1ot1 for its maintenance. To characterize the ncRNAs that mediate gene regulation through chromatin interaction we have isolated chromatin associated RNAs (CARs) from sucrose gradient fractioned chromatin. High-throughput sequencing of the CARs resulted in the identification of the 141 intronic and 74 intergenic regions harboring CARs. We characterized one of the intergenic CARs which regulate the transcription of the two neighboring genes by modulating the chromatin marks. In summary current thesis has uncovered unprecedented role of ncRNAs in gene expression via chromatin level regulation.

Indiana University-Purdue University Indianapolis (IUPUI) / The human genome generates a vast number of functionally and structurally diverse noncoding transcripts, incorporated into complex networks which modulate the activity of classic pathways. Long noncoding RNAs (lncRNA) have been shown to exhibit diverse regulatory roles in various physiological and pathological processes. Hypoxia, a key feature of the tumor microenvironment, triggers adaptive responses in cancer cells that involve hundreds of genes. While the coding component of hypoxia signaling has been extensively studied, much less information is available regarding its noncoding arm. My doctoral work identified and functionally characterized a novel hypoxia-inducible lncRNAs encoded from the miR193b-host gene (MIR193BHG) locus, on chromosome 16. In the pursuit of understanding how MIR193BHG responds to hypoxia, we discovered a more complex transcriptional control of MIR193BHG by hypoxia. Ectopic expression of MIR193BHG in breast cancer cell lines in vitro and in xenografts significantly represses cell invasion, as well as the metastasis to lung and liver. Conversely, inhibition of MIR193BHG promotes cancer cell invasiveness and metastasis. RNAseq followed by pathway analysis revealed that MIR193BHG is a negative modulator of cholesterol biosynthesis pathway. MIR193BHG exerts a highly coordinated effect on the expression of cholesterol biosynthetic genes which leads to a measurable impact on the total cellular cholesterol content. The role of MIR193BHG in cholesterol metabolism also provided a mechanistic explanation for the sex maturation associated SNPs located in vicinity of this gene locus. Our work also provided preliminary insights into the functional mechanism of MIR193BHG by showing that its modulation of genes in cholesterol synthesis is predominantly at transcriptional level. Overall, my dissertation project identified a non-canonical hypoxia-inducible lncRNA, MIR193BHG, which modulates breast cancer invasion and metastasis via finetuning of cholesterol synthesis.

Since its early discovery as a messenger, RNA has been shown to play a diverse set of regulatory, structural and even catalytic roles. The more recent understanding that the genome is pervasively transcribed stimulated the discovery of a new prevalent class of long non coding RNAs (lncRNAs). While these are lower abundant and relatively less conserved than other class of functional RNAs, lncRNAs are emerging as key players in different cellular processes in development and disease.

Eukaryotic genomes are pervasively transcribed and frequently generate long noncoding RNAs (lncRNAs). However, most lncRNAs remain uncharacterized. In this work, a set of positionally conserved intergenic lncRNAs in the fission yeast Schizosaccharomyces pombe genome are selected for further analysis. Deleting one of these lncRNA genes (ncRNA.1343) exhibited a clear phenotype: increased drug sensitivity. Further analyses revealed that deleting ncRNA.1343 also disrupted a previously unannotated lncRNA, termed nc-tgp1, transcribed in the opposite orientation of the predicted ncRNA.1343 gene and into the promoter of the phosphate-responsive permease gene tgp1+. Detailed analyses revealed that the act of transcribing nc-tgp1 into the tgp1+ promoter increases nucleosome density and prevents transcription factor access. Decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation, while nc-tgp1 loss induces tgp1+ in repressive phosphate-rich conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of nc-tgp1 transcription. Similarly, lncRNA transcription upstream of pho1+, another phosphate-regulated gene, increases nucleosome density and prevents transcription factor binding to repress pho1+ in phosphate-replete cells. Importantly, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription occurs in the absence of RNAi and heterochromatin components. Instead, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription resembles examples of transcriptional interference reported in other organisms. Thus, tgp1+ and pho1+ are the first documented examples of genes regulated by transcriptional interference in S. pombe.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19974号 / 薬科博第65号 / 新制||薬科||7(附属図書館) / 33070 / 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 馬見塚 拓, 教授 緒方 博之, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Recently, the discovery of noncoding RNAs (ncRNAs), such as long noncoding RNAs (lncRNAs) has altered the traditional view of gene regulation. Sequencing of genomes has brought to light the vast stretches of non-protein coding DNA regions that transcribe non-protein coding RNA. LncRNAs are multifunctional and extremely diverse. They can act as signals, decoys, scaffolds, guides, or enhancers. Several lncRNAs, such as Fendrr and Bvht, have been found to have important regulatory functions in cardiac disease and development. The Glt2-Dio3 locus, which is enriched in cardiac muscle, harbors two long intragenic RNAs, MEG3 and MEG8, and harbors one of the largest mammalian miRNA clusters. MEG3, which is termed Gtl2 in rat and mouse, contain 10 exons that are alternatively spliced and give rise to several variants. Gtl2 is conserved across human, rat, and mouse, which makes it an ideal candidate for research and a possible target for therapies. Based on the growing evidence for lncRNAs playing a role in cardiac muscle and our research on the Gtl2-Dio3 microRNAs (miRNAs), I focused on investigating the Gtl2 lncRNA in the heart. Antisense oligonucleotides (GapmeRs) were used to knockdown Gtl2 lncRNA expression levels in cultured, primary neonatal cardiomyocytes in basal and hypertrophic conditions. Although Gtl2 was effectively knocked down in basal conditions I was unable to achieve efficient knockdown in hypertrophic cardiomyocytes induced by phenylephrine treatment. Consequently, I did not observe any modulation of hypertrophy as determined by changes in the expression of Nppa and Nppb, established markers of cardiomyocyte hypertrophy.. Next, I utilized short hairpin RNA (shRNA) to knockdown Gtl2 lncRNA expression levels and obtained robust knockdown. Lastly, I designed a cardiac tropic adeno-associated virus 9 (AAV9) encoding MEG3 DNA for in vivo overexpression experiments as well as an adenovirus encoding MEG3 for in vitro overexpression experiments. These reagents will provide valuable resources for dissecting the functions of the Gtl2 lncRNA. Studies investigating the roles of Gtl2 in the diseased heart my lead to the development of other potential therapies to treat cardiac disease.