Fujii, H. | Search bioRxiv

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↗