Biology subjects

Fujita, T.

Publications and source records attributed to Fujita, T..

5 recordsLinked to original sources

bioRxiv2018

Transfer RNA Genes Affect Chromosome Structure and Function via Local Effects

The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs, these loci are also binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromosome folding or chromosome tethering. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long range HML-HMR heterochromatin clustering. We propose that the tDNAs primarily affect local chromatin structure that result in effects on long-range chromosome architecture.

molecular biology↗

bioRxiv2018

Role of the high-affinity leukotriene B4 receptor signaling in fibrosis after unilateral ureteral obstruction in mice

Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 receptor BLT1 during development of kidney fibrosis in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice with unilateral ureteral obstruction (UUO). We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of WT and BLT1-/- UUO mice. Accumulation of immunoreactive type I collagen in UUO kidneys of WT mice increased over time; however, the increase was less prominent in BLT1-/- mice. Accumulation of S100A4-positive fibroblasts also increased temporally in WT UUO kidneys, but was again less pronounced in those of BLT1-/- mice. The same was true of mRNA encoding transforming growth factor-{beta} (TGF)-{beta} and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-{beta}, also increased temporally in WT UUO and BLT1-/- kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-{beta}/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/--BM[->]WT UUO kidneys than in WT-BM[->]WT kidneys. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis.

pharmacology and toxicology↗

bioRxiv2018

Non-canonical role of E3 ligase RIPLET in innate immunity as a co-receptor for RIG-I

Ubiquitin (Ub) and its E3 ligases play diverse biological roles, from proteasomal degradation to innate immune signaling. The conventional view posits that E3 ligases function primarily through conjugating Ub to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET utilizes its dimeric structure and the bivalent binding mode to preferentially recognize RIG-I preoligomerized on dsRNA. RIPLET can also adopt an alternative binding mode for filamentous oligomers of RIG-I assembled on longer dsRNAs, cross-bridging RIG-I filaments in both ubiquitin-dependent and-independent manners. The resultant receptor clustering leads to the formation of aggregate-like cytosolic granules and the stimulation of RIG-I-mediated antiviral signaling in a RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. This study also highlights previously unrecognized mechanisms by which an E3 ligase induces receptor clustering and signal amplification, and offers insights into the unique cellular function of membrane-less granule assembly.\n\nOne-sentence summaryRIPLET functions as a molecular glue to assemble signaling scaffolds for the antiviral innate immune receptor RIG-I.

immunology↗

bioRxiv2017

Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis

We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules. In this study, we generated transgenic mice expressing 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-dCas9) and retrovirally transduced gRNA into primary CD4+ T cells from these mice for enChIP. Using this approach, we achieved high yields of enChIP at the targeted genomic region. Our novel transgenic mouse lines provide a valuable tool for enChIP analysis in primary mouse cells.

biochemistry↗

bioRxiv2016

Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line

Chromosomal interactions regulate genome functions, such as transcription, via dynamic chromosomal organization in the nucleus. In this study, we identified genomic regions that physically bind to the promoter region of the Pax5 gene in the chicken B-cell line DT40, with the goal of obtaining mechanistic insight into transcriptional regulation through chromosomal interaction. Using insertional chromatin immunoprecipitation (iChIP) in combination with next-generation sequencing (NGS) (iChIP-Seq), we found that the Pax5 promoter bound to multiple genomic regions. The identified chromosomal interactions were independently confirmed by in vitro engineered DNA-binding molecule-mediated ChIP (in vitro enChIP) in combination with NGS (in vitro enChIP-Seq). Comparing chromosomal interactions in wild-type DT40 with those in a macrophage-like counterpart, we found that some of the identified chromosomal interactions were organized in a B cell-specific manner. In addition, deletion of a B cell-specific interacting genomic region in chromosome 11, which was marked by active enhancer histone modifications, resulted in moderate but significant down-regulation of Pax5 transcription. Together, these results suggested that Pax5 transcription in DT40 cells is regulated by inter-chromosomal interactions. Moreover, these analyses showed that iChIP-Seq and in vitro enChIP-Seq are useful for non-biased identification of functional genomic regions that physically interact with a locus of interest.

molecular biology↗