Satoshi Ohta

The bcl-x gene, a bcl-2 family member, is highly regulated during lymphoid development, and its expression modulates apoptosis in lymphoid and other cell populations. Several forms of bcl-x mRNAs with different biologic functions have been described in rodents and humans. In this study, we have determined the organization and promoter region of the mouse bcl-x gene in an effort to understand the molecular basis for the different bcl-x mRNA species identified in tissues. We show that mouse bcl-x maps to the distal mouse chromosome 2 at approximately 89 cM, and exhibits a three-exon structure with an untranslated first exon and a facultative first intron. The coding region of bcl-xL is generated by the juncture of exons II and III through a splicing reaction, whereas bcl-xS is generated by an alternatively utilized donor splice site located within exon II. Analysis of multiple cDNAs and primer extension experiments revealed major transcription initiation sites in brain and thymus within a GC-rich region, with multiple Sp1-binding motifs located upstream of exon I. Another promoter was mapped to a 57-bp region localized upstream of the translation initiation codon by transfection of reporter constructs into FL5.12 and K562 cell lines. The remarkable similarity between the genomic regions of bcl-2 and bcl-x suggests that these genes have evolved from a common ancestral gene or through gene duplication.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase delta. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied. RESULTS: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA. CONCLUSION: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp.

The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a molecular platform that induces caspase-1 activation and subsequent IL-1β maturation, and is implicated in inflammatory diseases; however, little is known about the negative regulation of NLRP3 inflammasome activation. In this article, we identified an E3 ligase, Ariadne homolog 2 (ARIH2), as a posttranslational negative regulator of NLRP3 inflammasome activity in macrophages. ARIH2 interacted with NLRP3 via its NACHT domain (aa 220-575) in the NLRP3 inflammasome complex. In particular, we found that while using mutants of ARIH2 and ubiquitin, the really interesting new gene 2 domain of ARIH2 was required for NLRP3 ubiquitination linked through K48 and K63. Deletion of endogenous ARIH2 using CRISPR/Cas9 genome editing inhibited NLRP3 ubiquitination and promoted NLRP3 inflammasome activation, resulting in apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization, pro-IL-1β processing, and IL-1β production. Conversely, ARIH2 overexpression promoted NLRP3 ubiquitination and inhibited NLRP3 inflammasome activation. Our findings reveal a novel mechanism of ubiquitination-dependent negative regulation of the NLRP3 inflammasome by ARIH2 and highlight ARIH2 as a potential therapeutic target for inflammatory diseases.