Biology subjects

Hodaka Fujii

Publications and source records attributed to Hodaka Fujii.

3 recordsLinked to original sources

bioRxiv2016

Identification of physical interactions between genomic regions by enChIP-Seq

Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. However, the methods for confidently detecting physical interactions between genomic regions remain limited. Here, we demonstrate the feasibility of using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) in combination with next-generation sequencing (NGS) (enChIP-Seq) to detect such interactions. In enChIP-Seq, the target genomic region is captured by an engineered DNA-binding complex, such as a CRISPR system consisting of a catalytically inactive form of Cas9 (dCas9) and a single guide RNA (sgRNA). Subsequently, the genomic regions that physically interact with the target genomic region in the captured complex are sequenced by NGS. Using enChIP-Seq, we found that the 5HS5 locus, which regulates expression of the {beta}-globin genes, interacts with multiple genomic regions upon erythroid differentiation in the human erythroleukemia cell line K562. Genes near the genomic regions inducibly associated with the 5HS5 locus were transcriptionally up-regulated in the differentiated state, suggesting the existence of a coordinated transcription mechanism directly or indirectly mediated by physical interactions between these loci. Our data suggest that enChIP-Seq is a potentially useful tool for detecting physical interactions between genomic regions in a non-biased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

Molecular Biology↗

bioRxiv2015

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins

The clustered regularly interspaced short palindromic repeat (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed \"in vitro enChIP\" using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first demonstrate that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we show that this technology can be employed to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.

Biochemistry↗

bioRxiv2014

Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins

BackgroundComprehensive understanding of mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. We have developed the insertional chromatin immunoprecipitatin (iChIP) technology to isolate specific genomic regions retaining molecular interactions and identify their associated molecules. iChIP consists of locus-tagging and affinity purification. The recognition sequences of an exogenous DNA-binding protein such as LexA are inserted into a genomic region of interest in the cell to be analyzed. The exogenous DNA-binding protein fused with a tag(s) is expressed in the cell and the target genomic region is purified with antibody against the tag(s). In this study, we developed the iChIP system using recombinant DNA-binding proteins to make iChIP more straightforward.\n\nResultsIn this system, recombinant 3xFNLDD-D (r3xFNLDD-D) consisting of the 3xFLAG-tag, a nuclear localization signal, the DNA-binding domain plus the dimerization domain of the LexA protein, and the Dock-tag is used for isolation of specific genomic regions. 3xFNLDD-D was expressed using a silkworm-baculovirus expression system and purified by affinity purification. iChIP using r3xFNLDD-D could efficiently isolate the single-copy chicken Pax5 (cPax5) locus, in which LexA binding elements were inserted, with negligible contamination of other genomic regions. In addition, we could detect RNA associated with the cPax5 locus using this form of the iChIP system combined with RT-PCR.\n\nConclusionsThe iChIP system using r3xFNLDD-D can isolate specific genomic regions retaining molecular interactions without expression of the exogenous DNA-binding protein in the cell to be analyzed. iChIP using r3xFNLDD-D would be more straightforward and useful for analysis of specific genomic regions to elucidate their functions.

Biochemistry↗