Human Genome Meeting 2002

Shanghai International Conference Centre, Shanghai, China

April 15, 2002

11/86 (2: Chromosome structure and epigenetic mechanisms)

Oral and poster presentation

Allele-specific expression and DNA methylation analyses of the Hrh4 lying next to the imprinted gene Impact

Kohji Okamura1, Yoichi Yamada1,3, Yuriko Hagiwara-Takeuchi1, Yoshiyuki Sakaki1,2, and Takashi Ito3 1Hum. Genome Ctr., Inst. Med. Sci., Univ. Tokyo, Japan, 2Hum. Genome Res. Gr., RIKEN Genomic Sci. Ctr., Japan, 3Div. Genome Biol., Cancer Res. Inst., Kanazawa Univ., Japan

It is well known that imprinted genes often form clusters in mammalian genomes, which are regulated as certain chromosomal domains. For example, there are two famous imprinted regions on mouse chromosome 7. They correspond to human chromosome 11 and 15, and are related to Beckwith-Wiedemann and Prader-Willi/Angelman syndromes, respectively. Now that the human genomic sequence is about to be completed, it has become much easier to find novel imprinted genes or clusters, as is well exemplified by a recent discovery of a large imprinting cluster on mouse chromosome 12. On the other hand, some genes such as U2afbp-rs and Neuronatin are imprinted in solo or accompanying no other imprinted genes in their vicinity. It is elusive whether the underlying mechanism for such isolated imprinted genes is similar to the one for those forming clusters. The mouse Impact is a paternally-expressed gene identified in our unique allelic message display screening. Since Impact is the first and sole imprinted gene mapped to chromosome 18, it is not clear whether it is lying in a new imprinting cluster. Furthermore, since its human homolog IMPACT is not imprinted, it is interesting to investigate the allele-specific expression of the neighboring genes in both species. The human genome project has revealed several genes adjacent to IMPACT, including the human HRH4 which is mapped only 7-kb downstream from IMPACT. This gene has already been cloned and characterized. Fortunately, we have found one SNP in its coding region from a Japanese, which changes histidine to arginine. The RT-PCR product amplified from her peripheral blood leukocytes was directly sequenced. The result clearly indicated that HRH4 is expressed biallelically in her leukocytes, consistent with the biallelic expression of IMPACT. Next, we cloned its mouse homolog, which is also located just downstream of Impact, and determine the genomic DNA sequence covering these two genes. Allele-specific expression and DNA methylation status of mouse Hrh4 were examined using JF1/Msf (JF), C57BL/6J (B6), and their reciprocal F1 hybrids (JF × B6, B6 × JF). The results show that both maternal and paternal alleles are transcribed, and the promoter region is methylated on both chromosomes in spleen where this gene is preferentially expressed. Although we cannot exclude the possibility of imprinting of other neighboring genes, mouse Impact seems to be an isolated imprinted gene. If this is the case, Impact would serve as a nice model to elucidate the mechanism for this unique type of genomic imprinting.