Laboratory for Genome Exploration Research Group; RIKEN Genomic Sciences Center
Differentially expressed genes between parthenogenote and androgenote are good candidates for the identification of imprinted genes, which are specifically expressed either from the maternal or the paternal allele. To systematically search differentially expressed genes between parthenogenote and androgenote, we used RIKEN full-length mouse cDNA microarray (17K), using the parthenogenote and androgenote mRNA as probes.
Originally, our group has been working for mouse genome encyclopedia project for the past five years. Riken mouse genome encyclopedia is defined the comprehensive archives consisting of four components, mouse full-length cDNA clone bank, mouse full-length cDNA sequence database, chromosomal location of all transcripts and expression profile of all transcripts. For this purpose, we take the strategy with three phases, (1) collection of full-length cDNAs, (2) sequencing of full length cDNAs, and (3) mapping of these clones onto the chromosome. The phase l project is to collect mouse full-length cDNA clones from various tissues and developmental stages, classifying them based on their 3'-end sequence tags, and constructing the RIKEN non-redundant cDNA clone set. In the phase 2, we rearrayed these clones to construct non-redundant cDNA clone set and applied three sequencing strategies. So far, over 21,000 cDNA clones have been sequenced and highly functionally annotated, resulting in about 16000 unique sequence including 2200 and 560 new genes encoding new protein in any organisms and in mammalians, respectively.
Using the encyclopedia clone sets, we selected about 25 genes as imprinted candidates, including known imprinted genes, such as IgfII (Insulin-like-growth factor II), Snrpn (small nuclear ribonuclear protein) and Neuronatin. For some of the other genes, we have confirmed these results by RT-PCR, using parthenogenetic, gynecogenetic embryos and/or normal diploid tissues of two reciprocal F1 crosses between B6 and Jf1 mice. Among these clones, ZAC/PLAGL1 that is reported as a transcriptional factor with zinc finger motif, was contained.
Independently, we have been screened the imprinted methylation in the human genomic DNA. We compared parthenogenetic DNA from the chimeric patient FD and androgenetic DNA from hydatidiform. mole, using restriction landmark genome scanning for methylation (RLGS-M). This screening resulted in identification of two novel imprinted loci, one of which (NV149) we mapped to the TNDM region of 6q24. From analysis of the corresponding genomic region, it turned out that NV149 lies approximately 60 kb upstream of the ZAC/PLAGL1 gene. RT-PCR analysis was used to confirm that ZAC/PLAGL1 is expressed only from the paternal allele in a variety of tissues.
This gene is mapped onto 6q24 in the same region of transient neonatal diabetes (TNDM) whose phenotype is transmitted in imprinted manner, TNDM is known to result from upregulation of a paternally-expressed gene, in consistent with the paternally expression profile of ZAC. The paternal expression, map position, and known biological properties of ZAC/PLAGL1 make it highly likely that it is the TNDM gene. In particular ZAC/PLAGL1 is a transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), which is the most potent known insulin secretion stimulator and an important mediator of autocrine control of insulin secretion in the pancreatic islet. Thus, we propose two novel high-throughput screening system to screen imprinted genes. In combination with the mapping information, we could rapidly identify the imprinted disease genes.