Protein arginine methylation is an essential post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Type I PRMTs transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to the arginine residues and catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Type II PRMTs generate MMA and symmetric dimethylarginine (SDMA). PRMT-catalyzed methylation is involved in many biological processes and human diseases when dysregulated. As the predominant PRMT, PRMT1 catalyzes an estimated 85% of all protein arginine methylation in vivo. Nevertheless, the product specificity of PRMT1 remains poorly understood. A few articles have been published regarding the kinetic mechanism of PRMT1, yet with controversial conclusions. To gain more insights into the product specificity of PRMT1, we dissected the active site of PRMT1 and identified two conserved methionines (Met-48 and Met-155) significant for the enzymatic activity and the product specificity. These two methionines regulate the final product distribution between MMA and ADMA by differentially affecting the first and second methyl transfer step. Current data show that Met-48 also specifies ADMA formation from SDMA. To further understand the kinetic mechanism of PRMT1, we developed a double turnover experiments to conveniently assay the processivity of the two-step methyl transfer. Using the double turnover experiments, we observed that PRMT1-catalyzed dimethylation is semi-processive. The degree of processivity depends on the substrate sequences, which satisfies the controversy between the distributive or partially processive mechanisms previously reported. We are using transient kinetics and single turnover experiments to further investigate the mechanism of PRMT1. Interestingly, during these studies, we found that PRMT1 may incur oxidative damage and the histidine affinity tag influences the protein characteristics of PRMT1. These studies have given important insights into the product specificity and kinetic mechanism of PRMT1, and provided a strong foundation for future studies on PRMT1.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic inflammation is a major driver of tumor progression in over fifty percent of breast cancers. Tumors activate inflammatory processes by secreting factors that recruit and trigger inflammatory cells to release cytokines such as Interleukin 6 (IL-6). IL-6 stimulates the activity of signal transducers and activators of transcription 3 (STAT3), a transcription factor that has been extensively studied for its role in promoting breast cancer. Recently, downregulated HIPPO signaling was shown to drive the pro-growth effects of IL 6. Reduced HIPPO signaling allows for the nuclear translocation of transcriptional co-activator yes associated protein (YAP), implicating IL-6 in the co-activation of several transcription factors such as the TEADs that trigger pro growth programs. While IL-6/STAT3 stimulation has been shown to increase YAP activity, the mechanism driving this remains undocumented. The Angiomotins (Amots) are adapters of the HIPPO pathway that directly bind and regulate YAP activity. Molecular characterization of Amot transcriptional regulation unexpectedly revealed a single promoter controlling the expression of its two major isoforms: Amot 130 and Amot 80. Through immunofluorescent analysis, this study found that total Amot levels were elevated across multiple breast tumor subtypes and highest in samples with increased presence of stromal inflammatory cells. Further, the induction of total Amot expression by IL 6 was found to be essential for YAP dependent growth of breast cancer cells. The activation of Amot transcription by IL-6 was found to be through Specificity Protein 1 (Sp1), a transcription factor that is activated by STAT3. This work connects the activation of YAP1 by IL-6/STAT3 through the elevation of Amot expression by Sp1. Taken together, this explains a new avenue whereby breast cancer cells acquire enhanced oncogenic properties in response to inflammatory signaling.

Reprinted from Cancer Biomarkers, 15(4), Nakashima et al., Urine CXCL1 as a biomarker for tumor detection and outcome prediction in bladder cancer, 357-364, Copyright (2015), with permission from IOS Press. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19596号 / 医博第4103号 / 新制||医||1014(附属図書館) / 32632 / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 武田 俊一, 教授 川村 孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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This study examines the association between the extent of legal intensity in firms’ financial reports and their financial reporting quality. Firms’ financial reports contain embedded legal content arising from contracts and agreements underlying their operations. Accountants interpret the contract provisions and conditions to determine the timing and amounts recognized in financial reports. Legal content is technical and complex, creating a potential for misinterpretation and errors in accounting for transactions, thus adversely affecting financial reporting quality. Using legal tags in companies’ 10-K XBRL filings, I construct a measure of the legal intensity of financial reports. I document an inverse association between legal intensity of financial reports and financial reporting quality, proxied by accruals quality, analyst earnings forecast errors and dispersion, earnings informativeness, and accruals informativeness. The inverse association is driven by firm-specific legal content rather than common legal content. There is also some evidence that the inverse association is strengthened by high operational uncertainty and attenuated by accounting experts on the audit committee. Collectively, this study increases our understanding of challenges in translating legal content into financial reports and the benefit of audit committee accounting experts in this setting. / Business Administration/Accounting

The purposes of this study were to investigate effects of partial block periodized strength training on physical performance and to examine relationships between initial muscle strength measured with isometric mid-thigh pull (IMTP) and performance changes after 7 weeks of strength training. Seventeen collegiate male soccer players participated. Initial muscle strength was determined using IMTP while physical performance included 10 m and 20 m sprints and static vertical jump with a polyvinyl chloride pipe (SJ0), 20 kg barbell (SJ20), and barbell loaded to 40 kg bar (SJ40). Performance testing was performed at three points: before first week (baseline), fourth week (T1), and seventh week (T2). Statistically small to moderate changes were found from baseline to T2 in peak power (PP; < 0.001, ES = 0.49), net impulse (NI; < 0.001, ES = 0.49), peak velocity (PV; < 0.001, ES = 0.62), allometrically scaled PP (PPa; < 0.001, ES = 0.62) in SJ20 and jump height (JH) in SJ40 ( < 0.001, ES = 0.36). Moderate to large correlations were found between isometric peak force and the changes from baseline to T2 in SJ20 PP ( = 0.04, = -0.49), SJ20 PF ( = 0.03, = -0.52), PPa ( = 0.04, = -0.50), and SJ20 allometrically scaled peak force ( = 0.04, = -0.49). Properly structured strength training maximizes task-specific physical performance. Initial muscle strength negatively affects the magnitudes of adaptations to physical performance.

Cooperative self-assembly between transcriptional regulators (TRs) and DNA cis-regulatory motifs (CRMs) coordinates precise gene expression in eukaryotes. Complexes of multivalent TRs enable the formation of highly specific regulatory connections between otherwise weakly-interacting, low-specificity molecular components and can be used to engineer regulatory specificity in synthetic gene circuits in yeast. Circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by relying on the collective action of multiple TRs to program functional circuit connections. Experiments and mathematical models demonstrate that assembly-mediated regulatory connections mitigate circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. This naturally-inspired approach offers a simple, generalizable means for building evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications. Assemblies of cooperative TRs have also been found to facilitate the gene regulatory network rewiring that drives evolution. TRs that arise in new spatiotemporal regulatory contexts and cooperatively bind to ancestral TRs are stabilized and localized by their interaction, providing an opportunity for developing TR-CRM connections. The stability of the cooperative complex further allows for individual connections to be strengthened or weakened to meet regulatory requirements. Engineered cooperative regulation, paired with laboratory evolution experiments, offers a unique opportunity to elucidate the direct contribution of TR cooperativity to establishing new regulatory connections across evolutionary time. With design guided by a predictive theory of gene network evolution, cooperative circuits are poised for CRM evolution in longterm culture experiments at accelerated timescales compared to circuits without TR-TR cooperativity. In a cooperatively regulated circuit, random mutations to the DNA will be preserved if they initiate or improve a regulatory connection with a stabilized TR, and will be identified by DNA sequencing. This simplified synthetic approach in a controlled culture environment will elucidate the direct contribution of TR-TR cooperativity to network rewiring and evolution.

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