42Department of Cancer Biology

Division of Cancer Cell Biology
癌防御シグナル分野
Professor
Associate Professor
Assistant Professor
Project Assistant Professor

Makoto Nakanishi, M.D., Ph.D.
Atsuya Nishiyama, Ph.D.
Teh-Wei Wang, Ph.D.
Yoshimi Imawari, M.D., Ph.D.

教 授
准教授
助 教
特任助教

医学博士
博士（理学）
博士（理学）
博士（医学）

中西王
井西山
廻敦
徳良

真哉
偉美

There is some evidence that senescent cells play an important role in aging andhealthy lifespan. However, little is known about the molecular basis of aging-related
pathologies. Our research is focused on understanding the common pathologies
underlying a variety of aging-related diseases. Currently, we are interested in therole of p16-positive senescent cells in the age-dependent decline of various organfunctions and the mechanism of senescent cell accumulation with aging. In
addition, we are focusing on the mechanism underlying the accumulation ofabnormal proteins as a cause of aging. By understanding the degradation
mechanisms of misfolded proteins, we are promoting research on abnormal cellularfunctions caused by the accumulation of protein aggregates, especially in thepathogenesis of neurodegenerative diseases. We are also investigating themolecular link between DNA methylation and the maintenance of genome stability.

1. LONRF family proteins are bona fide protein
quality control ubiquitin-ligases
Dan Li, Teh-Wei Wang, Sae Aratani, Satoshi Kawakami, Satoshi Omori, Maho Tamatani, Manabu
Ozawa1, Keiko Nakanishi2, Chieko Konishi, Sayaka
Yamane, Yoshie Chiba, Yoichi Furukawa3, Kenta
Nakai4, Shoichi Shimada5, Yuji Yamanashi6, TakashiOkada7, Hideyuki Okano8, Tatsushi Toda9, Yoshikazu Johmura10, and Makoto Nakanishi:
1Laboratory of Reproductive Systems Biology, 3Division of Clinical Genome Research, 4Laboratory ofFunctional Analysis in silico, Human GenomeCenter, 6Division of Genetics, 7Division of Molecularand Medical Genetics, Center for Gene and CellTherapy, IMSUT. 2Department of Pediatrics, Central Hospital, and Department of Perinatology, Institute for Developmental Research, Aichi HumanService Center. 5Department of Neuroscience andCell Biology, Osaka University Graduate School ofMedicine. 8Department of Physiology, Keio University School of Medicine. 9Department of MolecularNeurology, Graduate School of Medicine, Universityof Tokyo. 10Division of Cancer and Senescence Biology, Cancer Research Institute, Kanazawa University
Many age-related diseases are causally linked tothe misfolding of proteins, and certain environmental
stresses can trigger the misfolding of mature proteins.
To prevent this, all cells have evolved protein quality
control (PQC) systems. These include translation control, molecular chaperone activity, and proteolytic
degradation by either the proteasome or autophagy.
For example, cleaved protein products generated bystalled ribosomes on defective mRNAs have beenfound to be targets for degradation by molecularpathways initiated at the ribosome. The protein will
inevitably be cleaved and is likely to be defective if
the ribosome cannot reach the correct termination codon. As a result, it may be advantageous for the cell todegrade such incomplete nascent chains. Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to pro-

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tein aggregates that are not diluted by mitosis.
Therefore, post-mitotic cells may have a specific protein quality control (PQC) system. The LONRF familyof proteins consists of three isozymes, LONRF1-3.
LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds andubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. lonrf2-/mice exhibit age-dependent TDP-43-mediated motor
neuron (MN) degeneration and cerebellar ataxia.
Mouse iPS cell-derived MNs lacking LONRF2 showed
reduced survival, shortening of neurites, and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in human ALS patients-derived MNs are rescued by ectopic expressionof LONRF2.
Lonrf1 was ubiquitously expressed in different tissues. Its expression in LSEC and Kupffer cells increased with age in the liver. Lonrf1high Kupffer cells
showed activation of regulatory pathways of peptidase activity. In normal and NASH liver, the activation of NF-κB and p53 pathways and the suppressionof IFNα, IFNγ, and proteasome signaling were detected in LSECs. During wound healing process, Lonrf1high/p16high fibroblasts showed activation of cell
growth and suppression of TGFβ and BMP signaling.
Lonrf1high/p16low fibroblasts exhibited activation ofWNT signaling. These results suggest that althoughLonrf1 does not appear to be associated with senescence induction and phenotypes, Lonrf1 may play akey role in linking oxidative damage responses andtissue remodeling during wound healing in different
modes in senescent and non-senescent cells.
2. M2 macrophage-derived TGF-β induces ageassociated loss of adipogenesis through progenitor cell senescence
Xinyi Zeng, Teh-Wei Wang, Yoshimi Imawari, Yuki
Okamura, Kiyoshi Yamaguchi1, Seira Hatakeyama1,Satoshi Yamazaki2, Eigo Shimizu3, Seiya Imoto3,
Yoichi Furukawa2, Yoshikazu Johmura4, Makoto
Nakanishi
1
Division of Clinical Genome Research, 2Division ofStem Cell Biology, 3Division of Health Medical Intelligence, 4Division of Cancer and Senescence Biology,Cancer Research Institute, Kanazawa University
Adipose tissue is an endocrine and energy storage
organ composed of several different cell types, including mature adipocytes, stromal cells, endothelial
cells, and a variety of immune cells. Adipose tissue
aging contributes to the pathogenesis of metabolic
dysfunction and is likely induced by crosstalk between adipose progenitor cells (APCs) and immune
cells, but the underlying molecular mechanisms remain largely unknown. In this study, we revealed thebiological role of p16high senescent APCs, and investigated the crosstalk between each cell types in theaged white adipose tissue. We performed the single-cell RNA sequencing (scRNA-seq) analysis on thep16high adipose cells sorted from aged p16-CreERT2/Rosa26-LSL-tdTomato mice. On the other hand, we conducted the time serial analysis on the age-dependentbulk RNA-seq datasets of human and mouse white
adipose tissues to infer the transcriptome alteration of
adipogenic potential within aging. We show that M2
macrophage-derived TGF-β induces APCs senescence which impairs adipogenesis in vivo. p16high senescent APCs increase with age and show loss of adipogenic potential. The ligand-receptor interaction
analysis reveals that M2 macrophages are the donorsfor TGF-β and the senescent APCs are the recipient.
Indeed, treatment of APCs with TGF-β1 induces senescent phenotypes through mitochondrial ROS-mediated DNA damage in vitro. TGF-β1 injection into
gonadal white adipose tissue (gWAT) suppresses adipogenic potential and induces fibrotic genes as wellas p16 in APCs. A similar gWAT atrophy is observedin cancer cachexia by APCs senescence. Our results
suggest that M2 macrophage-derived TGF-β induces
age-related lipodystrophy by APCs senescence. TheTGF-β treatment induced the DNA damage, mitochondrial ROS, and finally cellular senescence inAPCs.
3. CDCA7 is a hemimethylated DNA adaptor forthe nucleosome remodeler HELLSIsabel E. Wassing1, Atsuya Nishiyama, Moeri Hiruta2, Qingynuan Jia1, Reia Shikimachi2, Amika Kikuchi2, Keita Sugimura1, Xing Hong, Yoshie Chiba,
Chiristopher Jenness1, Kyohei Arita2, Hironori Funabiki1 and Makoto Nakanishi:
1
Laboratory of Chromosome and Cell Biology, TheRockefeller University2
Structural Biology Laboratory, Graduate School of
Medical Life Science, Yokohama City University
Mutations of the SNF2 family ATPase HELLS andits activator CDCA7 cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome,characterized by hypomethylation at heterochromatin. The unique zinc-finger domain, zf-4CXXC_R1, ofCDCA7 is widely conserved across eukaryotes but isabsent from species that lack HELLS and DNA methyltransferases, implying its specialized relation withmethylated DNA. Here we demonstrate that zf4CXXC_R1 acts as a hemimethylated DNA sensor.
The zf-4CXXC_R1 domain of CDCA7 selectively
binds to DNA with a hemimethylated CpG, but notunmethylated or fully methylated CpG, and ICF disesase mutations eliminated this binding. CDCA7 andHELLS interact via their N-terminal alpha helices,
through which HELLS is recruited to hemimethylated DNA. While placement of a hemimethylated CpG

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within the nucleosome core particle can hinder its recognition by CDCA7, cryo-EM structure analysis ofthe CDCA7-nucleosome complex suggests that zf4CXXC_R1 recognizes a hemimethylated CpG in themajor groove at linker DNA. Our study provides insights into how the CDCA7-HELLS nucleosome remodeling complex uniquely assists maintenanceDNA methylation.

Publications
1.		Li D, Johmura Y, Morimoto S, Doi M, Nakanishi K,Ozawa M, Tsunekawa Y, Inoue-Yamauchi A,Naruse H, Matsukawa T, Takeshita Y, Suzuki N,Aoki M, Nishiyama A, Zeng X, Konishi C, Suzuki
N, Nishiyama A, Harris SA, Morita M, Yamaguchi
K, Furukawa Y, Nakai K, Tsuji S, Yamazaki S,Yamanashi Y, Shimada S, Okada T, Okano H, Toda
T, Nakanishi M. LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes
late-onset neurological deficits. Nature Aging. 3:1001-1019, 2023.
2.		Miyashita R, Nishiyama A, Qin W, Chiba Y, Kori
S, Kato N, Konishi C, Kumamoto S, Kozuka-Hata
H, Oyama M, Kawasoe Y, Tsurimoto T, Takahashi
TS, Leonhardt H, Arita K, Nakanishi M. The termination of UHRF1-dependent PAF15 ubiquitin
signaling is regulated by USP7 and ATAD5. Elife.
e79013, 2023

3.		Li D, Wang TW, Aratani S, Omori S, Tamatani M,Johmura Y, and Nakanishi M. Transcriptomic
characterization of Lonrf1 at the single-cell level
under pathophysiological conditions. J Biochem.
173: 459-469, 2023
4.		Harris S A, Aratani S, Johmura Y, Suzuki N, Li D,Nakanishi M. In vivo dynamics of senescence inrhabdomyolysis-induced acute kidney injury. Biochem Biophys Res Commun. 673: 121-130, 2023
5.		Yabushita T, Chinen T, Nishiyama A, Asada S, Shimura R, Isobe T, Yamamoto K, Sato N, Enomoto Y,Tanaka Y, Fukuyama T, Satoh H, Kato K, Saitoh K,Ishikawa T, Soga T, Nannya Y, Fukagawa T, Nakanishi M, Kitagawa D, Kitamura T, Goyama S. Mitotic perturbation is a key mechanism of action ofdecitabine in myeloid tumor treatment. Cell Rep.
42:113098, 2023