Expression And Regulation Mechanism Analysis Of Blcap And Nnat During Mouse Development

There are two closely related imprinted gene, Bladder Cancer-Associated Protein(Blcap) and Neuronatin(Nnat), are located on mouse chromosome 2q F1. Nnat shows paternal allele-specific expression and is located in an 8.5 kb intron of Blcap gene, which predominatly maternally expressed in brain. Previous study found that this pair of imprinted gene may regulate with each other at the transcriptional level, but the mechanism is unclear. In addition, related research inferred that the hypomethylation of three potential CTCF binding sites 1.5 kb upstream of NNAT exon1 may lead to NNAT and BLCAP_V2a overexpression in Wilms tumor. Thus, this paper aims to study the expression patern of Blcap and Nnat during mouse development, their relationship at the transcriptional level and investigate the role of CTCF in their transcription regulation. Firstly, our research aims to give a systematic analysis on the expression patterns of Blcap and Nnat during mouse development by in situ hybridization(ISH) and q RT-PCR. The results of quantitative real-time PCR showed that the expression of Blcap and Nnat increased gradually from E9.5 to about E13 and reached the highest at E13.5 and E12.5, respectively, and then began to decrease until E18.5. Quantitative real-time PCR and RNA-seq analysis at E14.5, E15.5 and adulthood demonstrated that this pair of imprinted genes was predominantly expressed in brain compared with other tissues. ISH at E9.5, E11.5 and E15.5 showed that Blcap and Nnat were predominatly expressed in central nervous system, including forebrain, midbrain, afterbrain and diencephalon, in addition, we found Nnat was expressed more widely and plentiful than Blcap during this critical period of neurodevelopment, especially the noticeable Nnat signal in somite, fore limb, hind limb and pituitary body. Quantitative real-time PCR on different brain development stages of mouse showed that from E15.5 to P30, the expression level of Blcap in brain increased gradually, but Nnat expression was decreased rapidly. Then we focused on studying the dynamic expression of Blcap and Nnat during the mouse brain development by mean of ISH analysis on postnatal brain section to further understand the the expression profiles of Blcap and Nnat. As a result, Blcap and Nnat were widely and non-specific expressed in newborn mouse brain(P0). However, from P10 to P30, Blcap expression signal was specifically detected in the cerebellum, and olfactory bulb and hippocampus. And then the expression of Blcap decreased but was still be visible in the IGL of cerebellum, olfactory bulb and hippocampus at adulthood, different to the Blcap expression patern, Nnat transcript was still detected in P10 mouse whole brain. However, from P10 to adulthood, Nnat expression level was significantly down-regulated, and only weak signals could be detected in cerebellum and olfactory bulb. These results indicated that Blcap and Nnat act as an important role in neural system development, especially in the growth, maturity and function of cerebellum, olfactory bulb and hippocampus.Secondly, in order to preliminarily study the interplay of Blcap and Nnat, we designed the si RNA targeting these two imprinted genes, constructed the over-expression vector of Blcap and Nnat to transfect N2 a or NIH-3T3 cells. Results showed that Blcap expression level did not change after silencing and over expressing Nnat transcript. Similarly, silencing and over expressing Blcap transcript did not result in changing of Nnat, suggesting that there is no interplay between Blcap and Nnat at the transcriptional level.Lastly, we constructed CRISPR/Cas9 system targeting a region covering three core potential CTCF binding sites(CTCFBS#1~CTCFBS#3) upstream of Nnat promoter for exploring whether it acts as a putative imprinting control elements in Blcap/Nnat locu, which may contribute to regulating both Blcap and Nnat. Our result showed that the expression of Nnat was increased but not Blcap with disruption of these three core putative CTCF binding sites, indicating that there is a regulatory element within the Nnat locus that contains three potential CTCF binding sites may involve in the expression and regulation of Nnat but not Blcap.Taken together, this study has studied the expression patterns of Blcap and Nnat during mouse embryonic and postnatal development, preliminarily demonstrated that there may be no relationship between Blcap and Nnat at transcriptional, reported a potential Nnat regulation region within Blcap intron, which may contributed to the further investigation of the roles of Blcap and Nnat during mouse development and the regulation mechanism of imprinted gene.