Kobe University · Graduate School of Science and Technology Innovation

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• miR-4521-A novel biomarker for human lentigos.2024 · 2024年06月, Experimental dermatology, 33(6) (6), e15118, 英語, 国際誌
• GeF-seq: A Simple Procedure for Base-Pair Resolution ChIP-seq2024 · Springer US, 2024年07月, Methods in Molecular Biology, 39 - 53
• Apigenin Regulates Activation of Microglia and Counteracts Retinal Degeneration.Purpose: Photoreceptor degeneration is a major cause of blindness. Microglia are known to play key roles in the pathogenesis and progression of neural degeneration. We examined the possible use of apigenin, which is a naturally occurring flavonoid, for the treatment of photoreceptor degeneration through regulation of microglial activities. Methods: As in vitro analyses, BV2 and MG5 mouse microglia cell lines were stimulated in the presence or absence of apigenin, and their activation profile was examined. In vivo study was done using rd1 photoreceptor degeneration model, and apigenin was administered by intravitreal injection, and pathological feature was examined. Results: Cell survival was not affected by apigenin in either BV2 and MG5. Apigenin suppressed lipopolysaccharide (LPS)-induced chemokine production in both BV2 and MG5 cells, but phagocytosis was suppressed in MG5 cells but not in BV2 cells. Apigenin inhibited LPS-induced M1 activation but could not drive microglia toward the M2 phenotype. Apigenin suppressed the expression of miR-155 in a dose-dependent manner. Furthermore, the Ets protein level was suppressed by treatment of BV2 cells with apigenin. When rd1 mice were treated with apigenin by intravitreal injection, the expression of inflammatory chemokines in the retina was reduced, and activation of microglia and Müller glia was suppressed. Furthermore, the thickness of the outer nuclear layer of the retina of rd1 mice was thicker in apigenin-treated retinas. Conclusions: Taken together, local administration of apigenin to the retina is a potential therapeutic treatment for photoreceptor degeneration, which involves downregulation of microglia in the retina when photoreceptors are damaged.
• High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIP-seq.Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences.

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