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Endogenous Retroviral Insert Regulates Gene in Human Brain

Phenotypic traits of an organism are determined by the combination of protein coding sequences along with their regulatory elements, which generate tissue-specific expression profiles. In the recent research performed by two IBCh research groups, in collaboration with other Moscow institutes, it was described the impact of the human-specific endogenous retroviral insertion on regulation of expression of the gene important for proper CNS functioning. It was hypothesized that such interaction may have played a role in the human evolution by up-regulating the expression of this gene.

Suntsova Maria

Understanding the molecular basis of phenotypic differences between humans and their closest relatives — chimpanzee — can provide important clues to the human evolution as well as the etiology of several diseases. Considering high similarity of protein coding sequences between human and chimpanzee it is commonly thought that alterations in regulatory sequences (rather than genes themselves) are responsible for phenotypic differences between these two species. Due to high regulatory potential mobile elements, seem to be strong candidates to cause such alterations.

Scientists from the Group for Genomic Regulation of Cell Signaling Systems and Laboratory of molecular bases of embryogenesis (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow) and some other Moscow institutes, focused on studying endogenous retroviruses (ERVs) — self-reproducing DNA sequences that constitute significant part of the human genome. Endogenous retroviruses of the HERV-K (HML-2) family — one of four retroelement groups that were active at the moment of the human and chimpanzee divergence — drew special attention. Among HERV-K (HML-2) family members there are 133 copies that are human-specific. This group was chosen also because retroviral regulatory elements (e.g. promoter, enhancer and polyadenylation signal) are well studied and situated in specific regions, so called long terminal repeats (LTRs). These regulatory elements that are normally used by virus itself can also influence the expression of neighboring genes.

Six copies of human-specific ERVs located in close proximity of known human genes were chosen to study enhancer activity; the luciferase reporter assay was employed to probe ability of LTRs to activate promoters of neighboring genes. Three of them significantly activated expression of the reporter gene. And only one copy demonstrated correlation between the LTR enhancer activity and transcription level of the neighboring gene PRODH (see below). Additionally in the cells with the highest enhancer activity, endogenous LTR copy was hypomethylated. In other words, it was in active state, suggesting that enhancer effect observed in reporter experiments may also appear naturally resulting in high PRODH transcriptional activity. As you might have guessed, the chimpanzee promoter lacks retroviral insert, and using reporter assay it was demonstrated that the chimpanzee promoter was significantly weaker than that of human harboring retroviral enhancer.

Gene under the influence of the retroviral insert (PRODH) encodes prolinedehydrogenase — the key enzyme of proline catabolism. According to previous studies, PRODH activity is important for proper functioning of the central nervous system (CNS). Several PRODH mutations are associated with the susceptibility to schizophrenia and other psychiatric disorders. In CNS it is most probably involved in neuromediators synthesis, such as glutamate. PRODH transcriptional analysis demonstrated the highest expression level in the brain, especially in the hippocampus where LTR-enhancer was hypomethylated. Experiments with primary neuronal culture revealed that PRODH promoter together with the retroviral enhancer could drive neuron-specific expression of the reporter fluorescent gene. Thus, LTR acts as a tissue specific enhancer and is able to activate PRODH transcription in brain. It is worth to notice that average transcription level of PRODH in human brains is higher than in chimpanzee.

PRODH transcriptional regulation by the retroviral enhancer. Human-specific enhancer formed by a retroviral insert activates PRODH promoter in the brain. LTR enhancer activity is determined by interaction with SOX2 transcription factor.

Moreover, it was discovered that the LTR enhancer acts synergistically with the PRODH internal regulatory region — CpG-island. According to data from in vitro methylation experiments and methylation profiles in different tissues, methylation dramatically reduces LTR enhancer activity. In other words, when there is no use of LTR enhancer activity, it is usually “turned off” by methylation. Unlike the LTR, the CpG-island was hypomethylated in all studied samples independent of the PRODH transcription level. This indicates that the CpG “turn on” is important, but not sufficient for providing of high PRODH transcriptional activity.

To understand which transcription factors regulate the LTR enhancer activity, its sequence was searched for transcription factors binding motives; two functional SOX2 binding sites were discovered (see figure). Indeed, SOX2 addition led to amplifying of the LTR enhancer effect. Besides, SOX2 is highly expressed in hippocampus, where it can bind LTR.

In conclusion, we hypothesize that insertion of endogenous retrovirus near to PRODH may have increased its transcriptional activity in CNS that resulted in alteration of neuromediator balance. And this regulation may have played a role in human evolution.

Suntsova M., Gogvadze E.V., Salozhin S., Gaifullin N., Eroshkin F., Dmitriev S.E., Martynova N., Kulikov K., Malakhova G., Tukhbatova G., Bolshakov A.P., Ghilarov D., Garazha A., Aliper A., Cantor C.R., Solokhin Y., Roumiantsev S., Balaban P., Zhavoronkov A., Buzdin A. (2013). Human-specific endogenous retroviral insert serves as an enhancer for the schizophrenia-linked gene PRODH. Proc. Natl. Acad. Sci. U.S.A. 110, 19472–19477.

december 17, 2013