Sickle cell disease (SCD) is one of the most common hemoglobinopathies worldwide. It is caused by a homozygous mutation in codon 6 of the beta globin gene (HBB), which leads to polymerization of the variant hemoglobin and sickled red blood cells that obstruct blood vessels and reduce oxygen delivery to tissues. Patients with SCD have multiple clinical problems, including pain crises, anemia and organ damage. However, not all patients with SCD display all these clinical manifestations. One major factor for reduced occurrences of symptoms is fetal hemoglobin (HbF). HbF is the main hemoglobin in the fetus, and declines one year after birth to less than one percent of total hemoglobin. Nevertheless, there are individuals who continue to have high levels of HbF into adulthood, which is beneficial for an individual with SCD because HbF reduces the amount of sickle polymer in red blood cells. There are three major quantitative trait loci (QTL) associated with high HbF. However, these QTL account for 20-45% of HbF variance. Therefore, further investigation is required to fully understand how HbF is regulated. The HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23 is one of the major QTL associated with high HbF. This region is also known to regulate other erythroid-specific traits due to an enhancer element that promotes the expression of the downstream gene, MYB, which controls hemoglobin expression and erythroid proliferation and maturation. The presence of RNA polymerase II binding and a 50-bp transcript suggested that a long noncoding RNA (lncRNA) is transcribed from this region. LncRNAs are non-protein-coding transcripts greater than 200 nucleotides and are involved in gene regulation. Therefore, it was hypothesized that a lncRNA is transcribed from the enhancer of MYB and regulates hemoglobin expression. I characterized a novel lncRNA, 1283 bp in length that was differentially expressed among various tissue types, among erythroid progenitor cells with different hemoglobin makeup, and also during erythroid differentiation. Furthermore, knockdown of this lncRNA, named the HBS1L-MYB intergenic long noncoding RNA (HMI-LNCRNA), significantly increased HbF. Taken together, these observations suggest that HMI-LNCRNA can be a possible therapeutic target to increase HbF expression in patients with SCD and β-thalassemia. / 2018-05-01T00:00:00Z

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Non-coding antisense RNAs have been implicated in the epigenetic silencing of individual gene as well as chromosomal domains. While silencing of the overlapping gene by antisense RNAs has been well investigated, their functional role in silencing of chromosomal domains remains enigmatic. To elucidate mechanisms underlying the non-coding RNA mediated epigenetic silencing of chromosomal domains, we have chosen an antisense non-coding RNA, Kcnq1ot1, as a model system. Previously, a functional role of Kcnq1ot1 RNA and/or its transcriptional process has been implicated in silencing of multiple genes in the Kcnq1 imprinted cluster. However, these studies could not rule out the mechanisms involving other than Kcnq1ot1 RNA. Furthermore, it was also unclear how the Kcnq1ot1 promoter escapes silencing when its encoded RNA is capable of silencing flanking genes in cis. We have shown that NF-Y transcription factor plays a central role in the Kcnq1ot1 promoter activity, and that mutation of the NF-Y binding sites not only resulted in loss of silencing of flanking genes but also the ability of the Kcnq1ot1 promoter to protect against repressive chromatin marks, indicating that NF-Y maintains transcription-competent chromatin at the promoter through resisting the strong silencing effects of Kcnq1ot1 RNA. The Kcnq1ot1 RNA is an RNA Polymerase II encoded 91 kb long moderately stable nuclear transcript. We have demonstrated that it is the RNA not the act of transcription responsible for silencing and that the degree of silencing was proportional to the length of Kcnq1ot1 RNA. The kinetics of heterochromatin formation in relation to Kcnq1ot1 transcription revealed that overlapping gene was silenced initially by occlusion of basal transcription machinery and heterochromatin formation, whereas nonoverlapping gene was silenced subsequently by Kcnq1ot1-mediated heterochromatin spreading. This transcriptional silencing by Kcnq1ot1 RNA is mediated by an 890 bp region through promoting its interaction with the chromatin. Interestingly, we show that Kcnq1ot1 RNA establishes heterochromatin structures in a lineage-specific fashion by interacting with chromatin and chromatin remodelling complexes such as G9a and PRC2 complexes. More importantly, one of the parental chromosomes comprising Kcnq1 domain always found in the vicinity of perinucleolar region. Based on these data we proposed a mechanism whereby Kcnq1ot1 RNA establishes transcriptional silencing through recruitment of chromatin remodelling machinery and the maintenance of silencing achieved via targeting to the perinucleolar region.

Genomic imprinting is an epigenetic phenomenon that causes a subset of mammalian genes to be expressed from only one allele in a parent-of-origin manner. The defects in the imprinting regulation result in disorders that affect development, growth and metabolism. We have used the Kcnq1 imprinted cluster as a model to understand the mechanism of imprinted gene regulation. The imprinting at the Kcnq1 locus is regulated by a long noncoding RNA, Kcnq1ot1, whose transcription on the paternal chromosome is associated with the silencing of at least eight neighboring genes. By destabilizing Kcnq1ot1 in an episomal system, we have conclusively shown that it is the RNA and not the process of transcription that is required for the gene silencing in cis. Kcnq1ot1 RNA interacts with the chromatin modifying enzymes such as G9a and Ezh2 and recruits them to imprinted genes to establish repressive chromatin compartment and gene silencing. Using the episomal system, we have identified an 890 bp silencing domain (SD) at the 5’ end of Kcnq1ot1 RNA, which is required for silencing of neighboring reporter genes. The deletion of the SD in the mouse resulted in the relaxation of imprinting of ubiquitously imprinted genes (Cdkn1c, Kcnq1, Slc22a18, and Phlda2) as well as reduced DNA methylation over the somatic DMRs associated with the ubiquitously imprinted genes. Moreover, Kcnq1ot1 RNA interacts with Dnmt1 and recruits to the somatic DMRs and this recruitment was significantly affected in the SD mutant mice. By using a transgenic mouse, we have conditionally deleted Kcnq1ot1 promoter at different developmental stages and demonstrated that Kcnq1ot1 maintains imprinting of the ubiquitously imprinted genes by regulating DNA methylation over the somatic DMRs. Kcnq1ot1 is dispensable for the maintenance of repressive histone marks and the imprinting of placental-specific imprinted genes (Tssc4 and Osbpl5). In conclusion, we have described the mechanisms by which Kcnq1ot1 RNA establishes and maintains expression of multiple imprinted genes in cis.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, ranking the sixth most common cancer and third most common cause of cancer mortality worldwide. Alpha-fetoprotein (AFP) is a plasma protein that is highly expressed in the fetal liver and shut off after birth. AFP expression is elevated in regenerating adult liver and HCC and has been used extensively as a diagnostic marker of liver cancer. We have been studying mouse liver gene regulation to better understand mechanisms by which changes in gene expression contribute to liver development, homeostasis and disease. Zinc Fingers and Homeoboxes 2 (Zhx2) has been identified as a repressor of AFP, but the mechanism of this regulation remains unknown. Interestingly, all targets of Zhx2 that have been identified to date, including H19, Glypican 3, Elovl3 and Cytochrome P450 (CYP) genes, are also known to be misregulated in HCC. Thus, a better understanding of the mechanism by which these genes are regulated by Zhx2 will likely lead to new insights into gene regulation during HCC progression. Antisense transcripts belong to a diverse class of long noncoding RNA molecules > 200 nucleotides in length that often structurally resemble mRNAs, but do not encode proteins. While studying AFP mRNA regulation by Zhx2 in the mouse, our lab identified novel antisense AFP (asAFP) RNA transcripts that partially overlap the 3’ half of the mouse AFP gene. ENCODE tracks of ChIP-seq data for histone modifications in mouse liver show that the genomic region around the 5’ end of asAFP RNA has peaks for marks associated with promoters and enhancers. To better understand asAFP regulation, I identified the asAFP RNA 5’ end and the promoter elements that drive transcription. asAFP RNAs are ~5kb alternatively spliced, mainly cytoplasmic transcripts containing 2-4 exons. These transcripts were also detected in adult mouse liver RNA-seq data. asAFP is likely a noncoding RNA because it contains several small open reading frames that are 98 aa or smaller with no known functional domains or homology to known proteins. There is no evidence for similar transcripts in human liver. The abundance of asAFP RNA inversely correlates with AFP mRNA levels during postnatal liver development. Normally, asAFP RNA levels are high and AFP mRNA levels are low in the adult mouse liver. However, in the absence of Zhx2, AFP mRNA levels are higher and asAFP RNA levels are reduced, suggesting asAFP may be involved in the developmental regulation of AFP. Antisense transcripts function through a variety of mechanisms to positively or negatively regulate the expression of target genes. To explore the role of asAFP RNA in AFP gene regulation, I expressed segments of asAFP RNA in a mouse liver cell line and measured endogenous AFP mRNA levels. My data revealed that all segments of asAFP repressed endogenous AFP mRNA in trans. To determine the mechanism by which asAFP RNA regulates AFP, I expressed asAFP segments that overlapped only with exons or introns of AFP. The asAFP segments that overlap with the exons showed greater repression of endogenous AFP mRNA levels than those overlapping with intronic sequences. Additionally, I considered whether asAFP RNA repression of AFP mRNA may involve RNA editing by Adenosine deaminase acting on RNA (ADAR). ADARs convert adenosine to inosine in double-stranded RNAs that results in RNA degradation. My data indicate that AFP and asAFP dsRNA is not extensively edited, suggesting ADAR mediated decay is not involved in the regulation of AFP mRNA expression. However, further studies are required to determine the mechanism of cytoplasmic AFP mRNA degradation. Together, my data characterizes the transcriptional regulation of novel mouse asAFP transcripts and provides a model system to investigate how these transcripts regulate AFP mRNA through RNA-RNA interaction.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23060号 / 医博第4687号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 湊谷 謙司, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Covalent nucleotide modifications in RNAs affect numerous biological processes, and novel functions are continually being revealed even for well-known modifications. Among all RNA species, transfer RNAs (tRNAs) are highly enriched with diverse modifications, which are known to play roles in decoding and tRNA stability, charging, and cellular trafficking. However, studies of tRNA modifications have been limited in a small scale and performed by groups with different methodologies. To systematically compare the functions of a large set of noncoding RNA modifications in translational regulation, I carried out ribosome profiling in 57 budding yeast mutants lacking nonessential genes involved in tRNA modifications. Deletion mutants with enzymes known to modify the anticodon loop or non-tRNA substrates such as rRNA exhibited the most dramatic translational perturbations, including altered dwell time of ribosomes on relevant codons, and altered ribosome density in protein-coding regions or untranslated regions of specific genes. Several mutants that result in loss of tRNA modifications in locations away from the anticodon loop also exhibited altered dwell time of ribosomes on relevant codons. Translational upregulation of the nutrient-responsive transcription factor Gcn4 was observed in roughly half of the mutants, consistent with the previous studies of Gcn4 in response to numerous tRNA perturbations. This work also discovered unexpected roles for tRNA modifying enzymes in rRNA 2’-O-methylation, and in transcriptional regulation of TY retroelements. Taken together, this work revealed the importance and novel functions of tRNA modifications, and provides a rich resource for discovery of additional links between tRNA modifications and gene regulation.

Starting with the discovery of microRNAs and the advent of genome‐wide transcriptomics, non‐protein‐coding transcripts have moved from a fringe topic to a central field research in molecular biology. In this contribution we review the state of the art of “computational RNomics”, i.e., the bioinformatics approaches to genome‐wide RNA annotation. Instead of rehashing results from recently published surveys in detail, we focus here on the open problem in the field, namely (functional) annotation of the plethora of putative RNAs. A series of exploratory studies are used to provide non‐trivial examples for the discussion of some of the difficulties.

The 7SK small nuclear RNA (snRNA) is a key player in the regulation of polymerase (pol) II transcription. The 7SK RNA was long believed to be specific to vertebrates where it is highly conserved. Homologs in basal deuterostomes and a few lophotrochozoan species were only recently reported. On longer timescales, 7SK evolves rapidly with only few conserved sequence and structure motifs. Previous attempts to identify the Drosophila homolog thus have remained unsuccessful despite considerable efforts. Here we report on the discovery of arthropod 7SK RNAs using a novel search strategy based on pol III promoters, as well as the subsequent verification of its expression. Our results demonstrate that a 7SK snRNA featuring 2 highly structured conserved domains was present already in the bilaterian ancestor.

7SK RNA is a highly abundant noncoding RNA in mammalian cells whose function in transcriptional regulation has only recently been elucidated. Despite its highly conserved sequence throughout vertebrates, all attempts to discover 7SK RNA homologues in invertebrate species have failed so far. Here we report on a combined experimental and computational survey that succeeded in discovering 7SK RNAs in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. Despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. The additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3´ and 5´ stemloop motifs, and provide evidence for a third, structurally well-conserved domain.