THE EFFECT OF YY1 BINDING SITE TRUNCATION OF LINE1 ON ITS RETROTRANSPOSITION IN NEURONAL CELLS IN A MOUSE MODEL

Abstract Background Long interspersed nuclear element-1 (LINE1 or L1) is a major type of retrotransposons constituting about 20% of mammalian genomes, some of which still have retrotransposition ability. L1 is the only transposon that can retrotranspose itself using its own proteins. Although retrotransposition is strictly repressed by the host-defense mechanism, it is known that somatic L1 retrotransposition is transiently activated in early neural development. We previously reported the increased L1 copy number in the brains of patients as well as in animal models of psychiatric disorders (Bundo et al., Neuron 2014), suggesting the possibility that aberrant expression of L1 increases the risk for psychiatric disorders.L1 has its own internal promoter at the 5’ UTR. Several transcription factors that bind to the L1 5’ UTR play a critical role in the regulation of L1 expression. A YY1 binding site exists near the start of L1 at about +10 to +20. This site is shown to be important for the precise transcription start. Its effect on L1 expression, however, is controversial in studies using a teratocarcinoma cell line. One study showed that the promoter missing the site is 5 times less active, whereas another study found only a minor effect in a retrotransposition assay. Importantly, there are L1 copies missing the YY1 binding site in the human genome, and they are reported less methylated, evading the repression in the adult human brain. Thus, it is important to elucidate the effect of YY1 binding site on the L1 retrotransposition activity in the brain. Aims & Objectives In this study, we developed transgenic mice to investigate the effect of truncation of YY1 binding site on the L1 retrotransposition in the mouse brain. Method We utilized the L1EGFP construct for monitoring L1 retrotransposition. L1EGFP was made from human L1 and EGFP. In the system, the cells are labeled by EGFP expression only after the retrotransposition of the construct. We created 2 lines of L1EGFP mice by inserting intact or YY1 binding site-truncated L1EGFP (trL1EGFP) construct into the Rosa26 locus. We selected 5 mice from each line with the same sex and similar average age for this experiment. We perfused the mice and fixed the isolated brains overnight with 10% formalin neutral buffer solution. Then, the brain samples were placed in 30% sucrose solution and kept at -4°. The samples were sectioned with microtome and immunostained for EGFP. Results There was a considerable number of EGFP-labeled neurons in L1EGFP mice, mostly gathered in the neocortex and dentate gyrus. We also found EGFP-labeled neuroglia in the brain such as astrocytes. We also detected EGFP-labeled neurons in trL1EGFP mice. We compare the frequency and patterns of EGFP- positive cells between two strains and will present them on the poster. Discussion & Conclusion By analyzing the results, functions of the YY1 binding site on the L1 retrotransposition will be revealed. The trL1EGFP mice will be a useful tool to investigate the role of L1 retrotransposition in neurological disorders, psychiatric disorders, and aging in human. References Bundo, M. et al (2014) ‘Increased L1 Retrotransposition in the Neuronal Genome in Schizophrenia,’ Neuron, 81(2), pp. 306–313.