Normal B cell development and Pax5 expression in Thy28/ThyN1-deficient mice.

Thy28, also known as ThyN1, is a highly conserved nuclear protein. We previously showed that in a chicken mature B cell line, Thy28 binds to the promoter of the gene encoding Pax5, a transcription factor essential for B cell development, and positively regulates its expression. Here, we generated a Thy28-deficient mouse line to analyze its potential role in B cell development in mice. Thy28-deficient mice showed normal development of B cells, and the expression of Pax5 was comparable between wild-type and Thy28-deficient primary B cells. Thus, species-specific mechanisms regulate Pax5 expression and B cell development.

A refined two-step oligoribonucleotide interference-PCR method for precise discrimination of nucleotide differences.

We previously developed oligoribonucleotide (ORN) interference-PCR (ORNi-PCR), in which an ORN hybridises with a complementary DNA sequence and inhibits PCR amplification across the sequence in a sequence-specific manner. Suppression of target amplification by ORNi-PCR can be used to detect nucleotide differences such as mutations in a target sequence. In the present study, we refined the ORNi-PCR method and established a detailed technical protocol to precisely discriminate single-nucleotide differences. We first revealed that a two-step (denaturing and annealing plus elongation) rather than a standard three-step (denaturing, annealing and elongation) method is more suitable for stably hybridising an ORN to its target DNA sequence for sequence-specific suppression of target amplification. We then optimised the ORNi-PCR method using two-step cycles and established a step-by-step technical protocol. The optimised Two-Step ORNi-PCR method could discriminate single-nucleotide differences in genomic DNA or cDNA introduced by genome editing or mutations in cancer cells. In addition, we showed that Two-Step ORNi-PCR can detect the cancer cells possessing a single nucleotide mutation in a target locus mixed with a large number of cells harboring wild-type sequences in the locus so that the number of the cancer cells is only 0.2% of the total cell number. Two-Step ORNi-PCR is useful for simple, precise, cost-effective and positive detection of nucleotide differences in a wide range of molecular biology and medical applications.

Detection of genome-edited cells by oligoribonucleotide interference-PCR.

Genome editing by engineered sequence-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for analysis of gene functions. Several techniques have been developed for detection of genome-edited cells, but simple, cost-effective, and positive detection methods remain limited. Recently, we developed oligoribonucleotide (ORN) interference-PCR (ORNi-PCR), in which hybridization of an ORN with a complementary DNA sequence inhibits amplification across the sequence. Here, we investigated whether ORNi-PCR can be used to detect genome-edited cells. First, we showed that ORNs that hybridize to a CRISPR target site in the THYN1 locus inhibited amplification across the target site, but no longer inhibited amplification after the target site was edited, resulting in mismatches. Importantly, ORNi-PCR could distinguish even single-nucleotide differences. These features of ORNi-PCR enabled detection of genome-edited cells by positive PCR amplification. In addition, ORNi-PCR was successful in discriminating genome-edited cells from wild-type cells, and multiplex ORNi-PCR simultaneously detected indel mutations at multiple loci. However, endpoint ORNi-PCR may not be able to distinguish between mono- and bi-allelic mutations, which may limit its utility. Taken together, these results demonstrate the potential utility of ORNi-PCR for the screening of genome-edited cells.

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.

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 attempted to identify genomic regions that physically bind to the promoter region of the Pax5 gene, which encodes a master regulator for B cell lineage commitment, in a chicken B cell line, DT40, with the goal of obtaining mechanistic insight into transcriptional regulation through chromosomal interaction. We found that the Pax5 promoter bound to multiple genomic regions using locus-specific chromatin immunoprecipitation (locus-specific ChIP), a method for locus-specific isolation of target genomic regions, in combination with next-generation sequencing (NGS). 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 is regulated by B cell-specific inter-chromosomal interactions. Moreover, these analyses showed that locus-specific ChIP combined with NGS analysis is useful for non-biased identification of functional genomic regions that physically interact with a locus of interest.

A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells.

Accumulating evidence suggests that Pax5 plays essential roles in B cell lineage commitment. However, molecular mechanisms of B cell-specific expression of Pax5 are not fully understood. Here, we applied insertional chromatin immunoprecipitation (iChIP) combined with stable isotope labeling using amino acids in cell culture (SILAC) (iChIP-SILAC) to direct identification of proteins interacting with the promoter region of the endogenous single-copy chicken Pax5 gene. By comparing B cells with macrophage-like cells trans-differentiated by ectopic expression of C/EBPß, iChIP-SILAC detected B cell-specific interaction of a nuclear protein, Thy28/Thyn1, with the Pax5 1A promoter. Trans-differentiation of B cells into macrophage-like cells caused down-regulation of Thy28 expression. Loss-of-function of Thy28 induced decrease in Pax5 expression and recruitment of myosin-9 (MYH9), one of Thy28-interacting proteins, to the Pax5 1A promoter. Loss-of-function of MYH9 also induced decrease in Pax5 expression. Thus, our analysis revealed that Thy28 is functionally required for B cell-specific expression of Pax5 via recruitment of MYH9 to the Pax5 locus in chicken B cells.