Biology subjects

Nishiwaki, M.

Publications and source records attributed to Nishiwaki, M..

3 recordsLinked to original sources

bioRxiv2023

Single transcript-level metabolic responsive landscape of human liver transcriptome

Direct knowledge of gene regulation in human liver by metabolic stimuli could fundamentally advance our understanding of metabolic physiology. This information, however, is largely unknown, and this void is deeply rooted in a paradox that is caused by the inaccessibility of human liver to treatments and the insufficient annotation of liver transcriptome. Recent advances have uncovered immense complexity of transcriptome, i.e., multiple transcripts produced from one gene and extensive RNA modifications, which are all highly regulated and often condition-dependent. Establishing an inclusive annotation to study liver transcriptome dynamics thus requires human liver samples of diverse conditions, which are paradoxically unavailable due to the inaccessibility of human liver to treatments. In this work, we addressed these challenges by coupling an isogenic humanized mouse model with Nanopore single-molecule direct RNA sequencing (DRS). We first generated mice that carried humanized livers of identical genetic background, which were equivalent to clones of a single human liver, and then subjected the mice to representative metabolic treatments. We then analyzed the humanized livers with Nanopore DRS, which directly reads full-length native RNAs to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Thus, our system allows for constructing a de novo annotation of human liver transcriptomes reflecting metabolic responses and studying transcriptome dynamics in conjunction. Our analysis uncovered a vast number of novel genes and transcripts that have not been previously reported. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short read RNA-seq. We also revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between human and mouse and between two individuals using the liver-specific humanized mice. This revealed that transcriptome dynamics are highly species- and genetic background-dependent, which may only be faithfully studied in a humanized system that entails clones of the same human liver. Hence our work revealed a complex metabolic responsive landscape of human liver transcriptome and also provided a framework to understand transcriptome dynamics of human liver in response to physiologically relevant metabolic stimuli.

systems biology↗

bioRxiv2020

Multilevel integrative transcriptome analyses in humans and humanized mice define in vivo human lncRNA metabolic regulators

A growing number of long non-coding RNAs (lncRNAs) have emerged as vital metabolic regulators in research animals suggesting that lncRNAs could also play an important role in human metabolism. However, most human lncRNAs are non-conserved, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). As the sequence-function relation of lncRNAs has yet to be established, the identification of lncRNA metabolic regulators in animals often relies on their regulations by experimental metabolic conditions. But it is very challenging to apply this strategy to human lncRNAs because well-controlled human data are much limited in scope and often confounded by genetic heterogeneity. In this study, we establish an efficient pipeline to identify putative hLMRs that are metabolically sensitive, disease-relevant, and population applicable. We first progressively processed human transcriptome data to select human liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a metabolic disease population, and response to dietary intervention in a small disease cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and metabolic disease populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we were able to validate the regulatory role of one non-conserved hLMR in cholesterol metabolism. Mechanistically, this hLMR binds to an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. In summary, our study provides a pipeline to overcome the variabilities intrinsic to human data to enable the efficient identification and functional definition of hLMRs. The combination of this bioinformatic framework and humanized murine model will enable broader systematic investigation of the physiological role of disease-relevant human lncRNAs in metabolic homeostasis.

physiology↗

bioRxiv2019

Immortalization of human zone I hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

BackgroundHepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia.\n\nMethodsHepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10.\n\nResultsHepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings.\n\nConclusionsHepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

cell biology↗