We have found that three Tnfrsf genes present in the murine Kcnq1 domain, Tnfrsf23, 22 and 26, are expressed in embryos and are developmentally regulated. We also found that all three genes are expressed with a strong maternal bias. An antisense RNA, AK155734, is co-expressed and overlaps with Tnfrsf 22, and at least in neonatal heart, with Tnfrsf23. This non-coding RNA is imprinted in the same direction as the Tnfrsf genes.
In analyzing the evolutionary origin of Tnfrsf22
, we find that one Tnfrsf
gene is present within the Kcnq1
orthologous domain in non-mammalian vertebrates such as chicken and lizard. This suggests that one copy (at least) was also present in their common ancestor with mammals. Later in mammalian evolution, multiple duplications occurred. Although the scarcity of information in monotremes and marsupials does not allow us to determine when the first duplication took place, our data suggest that it occurred early during the evolution of mammals; we cannot, however, rule out that it occurred even earlier. What is clear is that after this initial duplication, Tnfrsf22/23
diverged. In some lineages, additional duplications occurred, while in others, (such as in primates) they appear to have been lost (). In mouse, the three Tnfrsf
genes are the result of two duplication events: the one that occurred before the split of euarchontoglires from laurasitherian mammals and a second duplication after the split of the mouse and rat lineages (). In fact, the Tnfrsf22
genes are located on two segmental duplications, as annotated in the UCSC genome browser (www.ucsc.edu
Figure 8. Model of the evolution of mouse and human Tnfrsf genes within the Kcnq1 region. Light gray line represents the species tree; thin black lines within represent the Tnfrsf gene tree. This figure represents only relevant lineages and species (more ...)
We conclude that these genes have been present in the Kcnq1
orthologous region of diverse vertebrates since before the establishment of imprinted expression in mammals ();6
genes did not initially acquire imprinting due to an insertion into a preexisting imprinted domain.
The Kcnq1 domain is regulated by a paternally expressed long non-coding RNA, Kcnq1ot1. Expression of Kcnq1ot1 leads to silencing of neighboring genes, with a range in the embryo that was assumed to be approximately half that of the placenta in the mouse (). The fact that the Tnfrsf genes exhibit imprinted expression suggests that they may be under the control of the Kcnq1ot1 RNA, although how the genes between Phlda2 and Tnfrsf26 escape repression will have to be investigated. There are other examples of escapees, such as Trpm5 and Tspan32 in the placenta, showing that silencing of genes is not uniform along the chromatin fiber. It is intriguing that the bias in expression is greatest in the Tnfrsf26 gene, the copy closest to the Kcnq1ot1 transcriptional unit. An alternative possibility is that there is an independent mechanism by which imprinting of the Tnfrsf genes is regulated. Several existing knockout mouse models will allow us to address this issue.
Neither of the Tnfrsf genes has a CG-rich promoter, so the mechanism of relative paternal repression may not be dependent on DNA methylation. Several of the genes in the domain have methylation-independent imprinted expression, but this is only true in the placenta. We cannot rule out that methylation marks on sequences that do not qualify as CG islands are important for imprinting at the Tnfrsf genes.
Interestingly, the antisense AK155734 gene has a similar expression pattern to the sense genes, albeit with a slightly later appearance. In addition, both have maternal bias, suggesting that either there is no transcriptional interference, or that they are expressed in distinct cells. Further experiments will be necessary to distinguish between these possibilities and to determine if AK155734 is functional.
No homolog of the murine Tnfrsf22, 23 and 26 genes had been identified in the human counterpart of the Kcnq1 domain to date, so we were interested in tracing the origin of this cluster. Our phylogenetic data showed that in fact, there are multiple Tnfrsf sequences in orthologous regions of many mammals, as well as in other vertebrates such as chicken and in lizard. In humans, there is a very short sequence with limited similarity to the murine Tnfrsf genes, which appears to have lost its function. Further studies will be needed to determine the selection regime (positive vs. purifying selection) operating during the evolution of this gene family within the Kcnq1 domain.
Duplicated loci are usually either maintained or lost during evolution, and if maintained, they can potentially serve as the raw material for neofunctionalization.7
New paralogs that are located in different genomic regions are more likely to have undergone adaptive evolution,8
and may acquire new regulatory signals and different expression patterns. Gene families that have rapidly expanded their copy number in mammals include those involved in immunity, such as the Tnf and Tnfr
superfamilies, with rapid gene gain and loss. The Tnfrsf
genes have been very dynamic during mammalian evolution with regards to species-specific gains and losses. For example, in rodents, the guinea pig lineage has undergone numerous expansions of Tnfrsf
, and the mouse has had a duplication after the split with rat. On the other hand, primates may have lost the genes within the Kcnq1
region altogether, with only a trace remaining in humans (). It is interesting to note that the murine Tnfrsf
genes lack cytoplasmic domains, suggesting they are snippets of original genes that were duplicated or relocated from other regions and can provide the substrate for expansion of their functions by adding different domains.
In conclusion, our results, in conjunction with the detailed biochemical studies previously reported,3
are suggestive of a developmental function for the Tnfrsf
genes in the mouse embryo, possibly acting as decoy receptors. The allele-specific studies show parent-of-origin biases in expression, although further studies are required to determine if the Kcnq1
imprinting control region or the Kcnq1ot1
non-coding RNA are implicated in these patterns. Furthermore, our phylogenetic analysis shows that Tnfrsf
genes were present within the Kcnq1
region before the establishment of imprinting.