Science & Technologies

DNA is associated with proteins and RNAs to form chromatin which regulates gene functions such as epigenetic regulation and transcription. Comprehensive understanding of chromatin and the mechanisms of such genome functions requires the identification of molecules that bind to the genomic regions of interest in vivo.

However, non-biased methods to identify the molecules bound to specific genomic loci in vivo are limited. To perform biochemical and molecular biological analyses of specific genomic regions, Epigeneron developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies that comprise insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChiP) to purify the genomic regions of interest.

Epigeneron is also implementing our own drug discovery research focusing on specific targets, using the locus-specific ChIP technologies, that regulate gene expression associated with the cause of diseases in oncology and neurology.

The mechanism of locus-specific ChIP is as follows:

  • Target genomic regions are tagged with engineered DNA-binding molecules (enChIP) or exogenous DNA-binding molecules recognizing their binding elements inserted into the target genomic regions (iChIP).
  • The resultant cells are stimulated and crosslinked with formaldehyde or other crosslinkers, if necessary.
  • The cells are lysed, and DNA is fragmented.
  • The complexes including the engineered DB are subjected to affinity purification, such as immunoprecipitation.
  • The isolated complexes retain molecules interacting with the genomic region of interest. Reverse crosslinking and subsequent purification of DNA, RNA, proteins, or other molecules allows the identification and characterization of these molecules.
  • Fujita H, Fujita T, Fujii H: Using the CRISPR System: Methods and Applications. The CRISPR Journal (2021) 4, 290-300

    The CRISPR Journal
  • Fujita T, Yuno M, Fujii H: An enChIP system for the analysis of bacterial genome functions. BMC Res. Notes (2018) 11, 387.

    BMC Res. Notes
  • Hamidian A, Vaapil M, von Stedingk K, Fujita T, Persson CU, Eriksson P, Veerla S, De Preter K, Speleman F, Fujii H, Påhlman S, Mohlin S: Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative role of HDX as a negative regulator. Biochem. Biophys. Res. Commun. (2018) 499, 291-298.

    PubMed
  • Fujita T, Yuno M, Fujii H: enChIP systems using different CRISPR orthologues and epitope tags. BMC Res. Notes (2018) 11, 154.

    BMC Res. Notes
  • Fujita T, Kitaura F, Oji A, Tanigawa N, Yuno M, Ikawa M, Taniuchi I, Fujii H: Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for locus-specific ChIP analysis. Genes Cells (2018) 23, 318-325.

    Genes Cells
  • Fujita T, Kitaura F, Yuno M, Suzuki Y, Sugano S, Fujii H: Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line. DNA Res. (2017) 24, 537–548.

    DNA Res.
  • Fujita T, Yuno M, Suzuki Y, Sugano S, Fujii H: Identification of physical interactions between genomic regions by enChIP-Seq. Genes Cells (2017) 22, 506-520.

    Genes Cells
  • Fujita T, Yuno M, Fujii H: Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus. Sci. Rep. (2016) 6, 30485.

    Sci. Rep.
  • Fujita T, Yuno M, Fujii H: Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells (2016) 21, 370-377.

    Genes Cells
  • Fujita T, Yuno M, Okuzaki D, Ohki R, Fujii H: Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing. PLoS One (2015) 10, e0123387.

    PLoS One
  • Fujita T, Kitaura F, and Fujii H: A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells. PLoS One (2015) 10, e0116579.

    PLoS One
  • Fujita T, and Fujii H: Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins. BMC Mol. Biol. (2014) 15, 26.

    BMC Mol. Biol.
  • Fujita T, and Fujii H: Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for locus-specific ChIP using CRISPR. PLoS One (2014) 9, e103084.

    PLoS One
  • Fujita T, Asano Y, Ohtsuka J, Takada Y, Saito K, Ohki R, and Fujii H: Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Sci. Rep. (2013) 3, 3171.

    Sci. Rep.
  • Fujita T, and Fujii H: Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. (2013) 439, 132-136.

    Biochem. Biophys. Res. Commun.
  • Fujita T, and Fujii H: Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain. Adv. Biosci. Biotechnol. (2012) 3, 626-629.

    Adv. Biosci. Biotechnol.
  • Fujita T, and Fujii H: Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS One (2011) 6, e26109.

    PLoS One
  • Hoshino A, and Fujii H: Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J. Biosci. Bioeng. (2009), 108, 446-449.

    PubMed
  • Fujita T, and Fujii H: Purification of specific DNA species using the CRISPR system. Biology Methods and Protocols (BIOMAP) (2019) 4 (1), bpz008.

    BIOMAP
  • Fujita T, and Fujii H: in vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (in vitro enChIP) using CRISPR ribonucleoproteins in combination with next-generation sequencing (in vitro enChIP-Seq) for the identification of chromosomal interactions. Bio-protocol (2017) 7 (22), doi: 10.21769/BioProtoc.2612.

    Bio Protoc.
  • Fujita T, and Fujii H: Isolation of specific genomic regions and identification of associated molecules by enChIP. JoVE (2016) issue 107, doi: 10.3791/53478.

    JoVE
  • Fujita T, and Fujii H: Biochemical analysis of genome functions using locus-specific chromatin immunoprecipitation technologies. Gene Regul. Syst. Bio. (2016) Suppl. 1, 1-9.

    Gene Regul. Syst. Bio.
  • Fujita T, and Fujii H: Applications of engineered DNA-binding molecules such as TAL proteins and the CRISPR/Cas system in biology research. Int. J. Mol. Sci. (2015) 16, 23143-23164.

    Int. J. Mol. Sci.
  • Fujii H, and Fujita T: Isolation of specific genomic regions and identification of their associated molecules by engineered DNA-Binding molecule-mediated chromatin immunoprecipitation (enChIP) using the CRISPR system and TAL proteins. Int. J. Mol. Sci. (2015) 16, 21802-21812.

    Int. J. Mol. Sci.
  • Fujita T, and Fujii H: Identification of proteins interacting with genomic regions of interest in vivo using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Bio-protocol (2014) 4 (10), doi: 10.21769/BioProtoc.1124.

    Bio Protoc.
  • Fujita T, and Fujii H: Locus-specific biochemical epigenetics/chromatin biochemistry by insertional chromatin immunoprecipitation. ISRN Biochem. (2013) 2013, 913273.

    ISRN Biochem.
  • Fujita T, and Fujii H: New directions for epigenetics: application of engineered DNA-binding molecules to locus-specific epigenetic research. Handbook of Epigenetics, Second Edition, Academic Press (Elsevier), Editor: Trygve O. Tollefsbol, (2017) 635-652.

  • Fujita T, and Fujii H: Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Methods Mol. Biol. (2015) 1288:43-52. doi: 10.1007/978-1-4939-2474-5_4.

    PubMed

Get In Touch