Yoshikazu Johmura

Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication

Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

Necessary and Sufficient Role for a Mitosis Skip in Senescence Induction

Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.

FAD24 Acts in Concert with Histone Acetyltransferase HBO1 to Promote Adipogenesis by Controlling DNA Replication

Preadipocytes differentiate into adipocytes through approximately two rounds of mitosis, referred to as mitotic clonal expansion (MCE), but the events early in the differentiation process are not fully understood. Previously, we identified and characterized a novel gene, fad24 (factor for adipocyte differentiation 24), induced to express at the early stages of adipocyte differentiation. Although fad24 clearly has crucial roles in adipogenesis, its precise functions remain unknown. Here we show that the knockdown of fad24 by RNAi in 3T3-L1 preadipocytes repressed MCE. Moreover, FAD24 interacts with HBO1, a histone acetyltransferase and positive regulator of DNA replication initiation. The knockdown of hbo1 repressed MCE and adipogenesis, indicating that FAD24 acts in concert with HBO1 to promote adipogenesis by controlling DNA replication. Regarding the molecular mechanisms behind the regulation of DNA replication by fad24, we revealed that FAD24 co-localizes with HBO1 to chromatin during late mitosis, which is when the prereplication initiation complex is assembled. Furthermore, chromatin immunoprecipitation experiments indicated that FAD24 localizes to origins of DNA replication with HBO1. When fad24 expression was inhibited during adipocyte differentiation, the recruitment of HBO1 to origins of DNA replication was reduced. Thus, FAD24 controls DNA replication by recruiting HBO1 to origins of DNA replication and is required for MCE during adipocyte differentiation.

The novel gene fad104, containing a fibronectin type III domain, has a significant role in adipogenesis

A novel gene named fad104 (factor for adipocyte differentiation‐104), whose expression level quickly increased in the early stage of adipogenesis, was isolated and characterized. The deduced amino acid sequence of fad104 revealed the possible presence of a fibronectin type III domain and transmembrane domain. The expression of fad104 was detected in adipocyte differentiable 3T3‐L1 cells but not observed in the non‐adipogenic cell line NIH‐3T3. Moreover, the ability of 3T3‐L1 cells to differentiate declined with the knockdown of fad104 by RNA interference, strongly indicating that fad104 functions as a positive regulator of adipogenesis.

SLC39A14, a LZT protein, is induced in adipogenesis and transports zinc

During adipocyte differentiation, there is an underlying complex series of gene expressions. We have previously isolated many genes whose expression levels are quickly elevated by the addition of inducers to mouse 3T3‐L1 preadipocyte cells. Here we report the isolation and characterization of SLC39A14, a member of the LZT proteins, one of the subfamilies of ZIP transporters. The expression of the SLC39A14 gene was strongly and rapidly induced at the early stages of differentiation. Moreover, it was highly restricted to the potential differentiation state of 3T3‐L1 cells and the expression level was quite low in the nonadipogenic NIH‐3T3 cells, indicating a dominant expression in adipocyte differentiation. The zinc uptake assay revealed that SLC39A14 functions as a zinc transporter. Taken together, these results suggest that SLC39A14 plays a role as a zinc transporter during the early stages of adipogenesis.

Fad24, a mammalian homolog of Noc3p, is a positive regulator in adipocyte differentiation

Adipocyte differentiation is controlled by complex actions involving gene expression and signal transduction. From metaphase to anaphase, peroxisome proliferator-activated receptor γ, the CCAAT/enhancer-binding protein family and sterol regulatory element-binding protein-1 are known to function as master regulators. However, the mechanism underlying the earliest step, which triggers the initiation of differentiation, remains unknown. In previous reports, we have isolated a number of genes, whose expression increases in the early stage of differentiation in the mouse 3T3-L1 preadipocyte cell line. Here we report the cloning of the full-length cDNA and characterization of an unknown gene isolated previously and named fad24 (factor for adipocyte differentiation 24). Fad24 encodes a protein consisting of 807 amino acids. The deduced amino acid sequence was shown to have a basic leucine zipper motif and a NOC domain. Expression of fad24 was rapidly induced after stimulation with inducers. Furthermore, overexpression of fad24 in NIH-3T3 cells promoted adipogenesis in the presence of a ligand for peroxisome proliferator-activated receptor γ. FAD24 localizes in the nucleus, especially within nuclear speckles. As the nuclear speckle functions as a nascent transcription and pre-mRNA splicing machinery, there is a possibility that FAD24 functions as one of the components for transcription and/or pre-mRNA splicing and positively regulates adipocyte differentiation.

Increased protein stability of CDKN1C causes a gain-of-function phenotype in patients with IMAGe syndrome.

Mutations in the proliferating cell nuclear antigen (PCNA)-binding domain of the CDKN1C gene were recently identified in patients with IMAGe syndrome. However, loss of PCNA binding and suppression of CDKN1C monoubiquitination by IMAGe-associated mutations hardly explain the reduced-growth phenotype characteristic of IMAGe syndrome. We demonstrate here that IMAGe-associated mutations in the CDKN1C gene dramatically increased the protein stability. We identified a novel heterozygous mutation, c.815T>G (p.Ile272Ser), in the CDKN1C gene in three siblings manifesting clinical symptoms associated with IMAGe syndrome and their mother (unaffected carrier). PCNA binding to CDKN1C was disrupted in the case of p.Ile272Ser, and for two other IMAGe-associated mutations, p.Asp274Asn and p.Phe276Val. Intriguingly, the IMAGe-associated mutant CDKN1C proteins were fairly stable even in the presence of cycloheximide, whereas the wild-type protein was almost completely degraded via the proteasome pathway, as shown by the lack of degradation with addition of a proteasome inhibitor, MG132. These results thus suggested that the reduced-growth phenotype of IMAGe syndrome derives from CDKN1C gain-of-function due to IMAGe-associated mutations driving increased protein stability.

SCF Fbxo22 -KDM4A targets methylated p53 for degradation and regulates senescence

Recent evidence has revealed that senescence induction requires fine-tuned activation of p53, however, mechanisms underlying the regulation of p53 activity during senescence have not as yet been clearly established. We demonstrate here that SCFFbxo22-KDM4A is a senescence-associated E3 ligase targeting methylated p53 for degradation. We find that Fbxo22 is highly expressed in senescent cells in a p53-dependent manner, and that SCFFbxo22 ubiquitylated p53 and formed a complex with a lysine demethylase, KDM4A. Ectopic expression of a catalytic mutant of KDM4A stabilizes p53 and enhances p53 interaction with PHF20 in the presence of Fbxo22. SCFFbxo22-KDM4A is required for the induction of p16 and senescence-associated secretory phenotypes during the late phase of senescence. Fbxo22−/− mice are almost half the size of Fbxo22+/− mice owing to the accumulation of p53. These results indicate that SCFFbxo22-KDM4A is an E3 ubiquitin ligase that targets methylated p53 and regulates key senescent processes.

Multiple facets of p53 in senescence induction and maintenance

Cellular senescence is a state of durable cell cycle arrest with metabolic activities distinct from those of the proliferative state. Since senescence was originally reported to be induced by various genotoxic stressors, such as telomere erosion and oncogenic signaling, it has been proposed to play a pivotal role in aging‐related changes and as an antitumorigenic barrier in vivo. However, the mechanisms underlying its induction and maintenance remain entirely elusive. We have recently found that abrupt activation of p53 at G2 results in a cell skipping mitosis and subsequently undergoing senescence. Surprisingly, we have also found that downregulation of p53 by SCFFbxo22 is crucial for the induction of a senescence‐associated phenotype. In this review, we provide an overview of recent advances in understanding the mechanisms underlying the timing and magnitude of activation of p53 during senescence.

CBP-93872 Inhibits NBS1-Mediated ATR Activation, Abrogating Maintenance of the DNA Double-Strand Break–Specific G2 Checkpoint

CBP-93872 was previously identified as a G2 checkpoint inhibitor using a cell-based high-throughput screening system. However, its molecular actions as well as cellular targets are largely unknown. Here, we uncovered the molecular mechanisms underlying abrogation of the G2 checkpoint by CBP-93872. CBP-93872 specifically abrogates the DNA double-stranded break (DSB)-induced G2 checkpoint through inhibiting maintenance but not initiation of G2 arrest because of specific inhibition of DSB-dependent ATR activation. Hence, ATR-dependent phosphorylation of Nbs1 and replication protein A 2 upon DSB was strongly suppressed in the presence of CBP-93872. CBP-93872 did not seem to inhibit DNA-end resection, but did inhibit Nbs1-dependent and ssDNA-induced ATR activation in vitro in a dose-dependent manner. Taken together, our results suggest that CBP-93872 is an inhibitor of maintenance of the DSB-specific G2 checkpoint and thus might be a strong candidate as the basis for a drug that specifically sensitizes p53-mutated cancer cells to DSB-inducing DNA damage therapy.