Our study determined that Pon1
exhibits preferential allelic expression in the liver and that the allelic expression level can change in a dynamic manner throughout embryonic development and is also dependent on genetic background. The allelic expression pattern of Pon1
was examined by Ono et al
), but only in neonatal liver and lung tissues. The allelic difference we detected in gene expression changed throughout development in a strain-dependent manner and was found to be biased against the allele carried by CAST and JF1, although the bias was stronger in crosses involving CAST and when the BL6 allele was maternally inherited. Our observations illustrate the first case of a developmentally regulated dynamic allelic expression pattern. A recent study performed by Wilkins et al
) demonstrated that differences in allelic expression can occur between different regions of a single-tissue sample from the same individual. Consequently, owing to the fact that whole-liver samples were used in our study, its findings may reflect differences in allelic expression occurring in a subset of cells within the liver.
An analysis of the human PON1 protein in infants revealed an increased expression level of 2 to 7-fold from birth until 6 to 12 months, at which point it reached a plateau (31
). The equivalent stage is reached at 3 weeks of age in mice and rats. We found that Pon1
transcript levels increased substantially during embryonic formation, providing evidence that Pon1 protein activity escalates during hepatic development and plateaus at postnatal stages. Future studies may determine whether such an increase in expression correlates with Pon1 enzymatic activity.
Monoallelic and preferential expression of human PON1
was observed in various liver and pancreatic foetal samples, yet we were unable to determine the parent-of-origin. The differences in human PON1
expression may be attributed to polymorphic imprinting, which has been observed in several human genes (32
). However, it is important to note that preferential monoallelic expression can occur in a non-parent-of-origin pattern (10
), as was seen in Pon1
at 12.5 dpc in BJ and JB hybrids (Fig. ). Consequently, it is plausible to hypothesize that PON1
is expressed monoallelically at early gestational stages, preferentially later in development, and biallelically expressed neonatally. Such a dynamic pattern of expression would account for the preferential and biallelic expression patterns seen in several of the human samples. However, the differences in PON1
expression may also be due to polymorphisms in cis
-acting regulatory regions between the biallelically and monoallelically expressed samples.
Variation in allelic expression dependent on developmental stage has been suggested to be a regulatory mechanism in the decline of lactase activity in mammalian maturation. Cis
-acting regulatory variants have been identified upstream of the lactase gene, and these have been associated with hereditary lactase persistence (34
). These variants have been associated with increased/decreased levels of lactase transcript and have been shown to exhibit differential binding of nuclear proteins (36
). Human studies have also shown a decline in the expression from the allele associated with hypolactasia with age (38
). Such a developmentally regulated pattern of preferential allelic expression may also occur in Pon1
, where the expression from one allele varies with developmental stage, due to differential binding of transcription factors.
Two common coding polymorphisms have been identified in human PON1, L55M and Q192R, where the former has been associated with a greater production of mRNA (L allele) (22
) and greater serum paraoxonase levels (18
). Brophy et al
) found that this polymorphism is in linkage disequilibrium with a polymorphism in the promoter region (–108C/T). To determine whether sequence variations in regulatory regions may likewise account for the preferential pattern of the expression observed in mice, we sequenced the 1 kb region upstream from the coding region and identified two SNPs. In silico
analysis of the SNPs identified a putative binding site for TFIID, a TATA-box-binding protein required for RNA polymerase II activity, at SNP –227, implicating a possible role for this general transcription factor in the expression pattern of Pon1
. However, this analysis does not exclude the possibility that cis
-acting regulatory elements may be located in other regions of the gene, including introns and 3′ regulatory regions.
An analysis of IL10
production and promoter cis
-acting variations within the locus identified specific haplotypes which were associated with higher allelic transcription (13
). In a similar analysis, an intronic SNP in the lymphotoxin-α gene was found to be correlated to the protein’s production owing to the haplotype-specific binding of a bHLH protein which gave rise to allele-specific regulation (39
). Such variations may account for the difference in Pon1
gene expression levels seen between JF1 and CAST mice strains. Further analyses using reporter-based promoter studies may corroborate the impact of cis
-acting variations on Pon1
The results of this study emphasize the importance of determining expression levels, not only in reciprocal crosses as shown here (3
), but also at different developmental stages when analysing preferential patterns of expression. This is highlighted by the fact that each gene in the paraoxonase family shows a different pattern of expression, although located within a cluster of imprinted genes. Such a finding could only be proved by reciprocal analyses of hybrid samples, which allows one to distinguish between monoallelic or preferential expression and true imprinting which must be parent-of-origin specific. In addition, our results were duplicated on an independent set of liver samples, further stressing the absence of random expression.
Our findings further stress the need to determine allelic levels at disease loci, since differences in these levels are sources of phenotypic variation in human genetic diseases. If the mechanism of Pon1 regulation is conserved, it may imply that polymorphisms in human PON1 may not be a clear indicator of increased risk for atherosclerosis, since the expression of both alleles is neither equivalent nor static. However, further experiments need to be performed to determine levels of human PON1 allelic variance.