In the Footsteps of the Rugged Trail of Transcription

The nature of life is intrinsically simple and complex, with merely four basic nucleotides that ultimately make up countless lives with distinct appearances. Darwin’s theory of evolution reveals how time shapes the inhabitants of the planet, whereby it is the fundamental components that determine who we are. It is not until several decades ago, where we finally cracked the mystery of the DNA molecule with the swirling structure. Since then, waves of biology studies have entered an exponential phase. With the development of technologies, scientists have increased their understanding of the mechanisms behind biological questions in various organisms. As the initial step of all biological processes, transcription from DNA to RNA has been extensively characterized. We have acknowledged that this series of action is not at all effortless, but largely regulated. To name a few, the state of chromatin structure, level of transcription factor binding, initiation, and elongation of transcription, the post-transcriptional regulation of messenger RNA, and not to mention the influence of non-coding RNA. Moreover, the messenger RNA is stepwisely exported to the cytoplasm of the cells and translated into protein in mammalian cells. Despite being located in two spatially separate compartments, the crosstalk is never ignored. Hence, it is of great interest to investigate whether this crosstalk between transcription and translation has any consequences to cellular functions. Our study looks at the starting point of transcription – promoter, by performing an unbiased screen for candidate promoters that could potentially affect the translation process Interestingly, we discovered a set of promoters with TATA box, which boosts the translation of messenger RNA. Using CRISPR-Cas9 genome editing, genome-wide analysis, and in vitro experiments, we found that the effect is not limited to promoters with TATA box. Instead it is a rather general mechanism dictated by the rate of transcription, more specifically transcription elongation. More importantly, the performance requires cap-dependent translation. To examine the responsible factor, we assess the level of RNA modification - m6A on the messenger RNA and observe a reverse correlation between the status of translation and the level of m6A modification. Notably, although m6A are mostly found around stop codons, in our study m6A modification within the coding region of genes are crucial for the suppressive effect on translation. Finally, we propose a model that co-transcriptional deposition of m6A via retarded RNA Pol II elongation could lead to translational control. The study opens new doors to grasping the hidden regulators of gene expression. The advancement of CRISPR-Cas9 genome editing has led to a myriad of genome- wide studies in various organisms. While most studies focus on the coding regions of the genome, we have previously established a systemic approach to study the functions of non-coding genome by targeting transcription factor binding sites at enhancer elements. To broaden our understanding about how enhancer elements coordinate gene expression, we performed a genetic screen of enhancers with AP1 binding motif in oncogene-induced senescence. We successfully validated two sgRNAs targeting the same AP1 motif, showing it can affect the process of senescence. Mutations of the core AP1 binding site, in turn, abolished the enhancer function. Later, it was discovered that the FOXF1 gene was the interacting target, where disruption of FOXF1 lead to similar effects on senescence. Together, we suggest that AP1, a downstream target of mThis thesis explores multiple aspects of the transcription regulatory network with regards to human cells - From the initiation of transcription to the efficiency of translation. _Chapter 2_ attempts to establish a connection between the transcription and translation processes. By employing multiple in vitro experiments, a positive correlation between transcription and translation was first discovered, where it was solely controlled via transcription rate. The study leads to the identification of one of the first known functions of m6A at coding regions. While seemingly counterintuitive to the current recognition about m6A on translation, we augment the role of m6A on translation via the discovery of its co-transcriptional deposition. _Chapter 3_ reviews the connection between the transcription steps of splicing, export, decay, and translation of mRNA, reinforcing the hypothesis that transcription is not an independent event, and is instead linked to the entire life cycle of mRNA. _Chapter 4_ investigates the functional role of AP1 in enhancer regions during OIS. The study results in the identification of a novel enhancer with an AP1 binding motif regulating senescence through its target gene FOXF1. Although extensively characterized, AP1 and FOXF1 are not reported to act as regulators of senescence. A new trans-regulatory network of genes to counterbalance the effect of oncogene activation was uncovered. In _Chapter 5_, a general discussion about the current view of the field is conducted. Finally, some outlooks are raised to potentially generate a better understanding of transcriptional regulation

Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation

Transcription and translation are two main pillars of gene expression. Due to the different timings, spots of action, and mechanisms of regulation, these processes are mainly regarded as distinct and generally uncoupled, despite serving a common purpose. Here, we sought for a possible connection between transcription and translation. Employing an unbiased screen of multiple human promoters, we identified a positive effect of TATA box on translation and a general coupling between mRNA expression and translational efficiency. Using a CRISPR-Cas9-mediated approach, genome-wide analyses, and in vitro experiments, we show that the rate of transcription regulates the efficiency of translation. Furthermore, we demonstrate that m6A modification of mRNAs is co-transcriptional and depends upon the dynamics of the transcribing RNAPII. Suboptimal transcription rates lead to elevated m6A content, which may result in reduced translation. This study uncovers a general and widespread link between transcription and translation that is governed by epigenetic modification of mRNAs

Do Credit Associations Compete with Each Other in Japanese Regional Lending Markets?

This paper examines whether credit associations in Japanese regional lending markets compete on price now that Japanese financial authorities have replaced the convoy system of financial regulation with the principle of competition. Specifically, the effects of the market share of credit associations in regional markets on their lending rates are empirically investigated. Accordingly, we determined that credit associations compete with each other in regional lending markets by using two different proxies for the market share held by credit associations in a region. The first proxy was the credit associations’ share of all deposits in a region and the second was the credit associations’ share of all branch offices in a region. In addition, credit associations that face more intense competition from regional banks in regional markets were found to face more intense competition from other credit associations

LncRNA-OIS1 regulates DPP4 activation to modulate senescence induced by RAS

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role

A comprehensive enhancer screen identifies TRAM2 as a key and novel mediator of YAP oncogenesis

Background: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions. Results: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopi

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of diseas

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease