L1 retrotransposition in neurons is modulated by MeCP2

Abstract

Long interspersed nuclear elements-1 (LINE-1 or L1s) are abundant retrotransposons that comprise approximately 20% of mammalian genomes. Active L1 retrotransposons can impact the genome in a variety of ways, creating insertions, deletions, new splice sites or gene expression fine-tuning. We have shown previously that L1 retrotransposons are capable of mobilization in neuronal progenitor cells from rodents and humans and evidence of massive L1 insertions was observed in adult brain tissues but not in other somatic tissues. In addition, L1 mobility in the adult hippocampus can be influenced by the environment. The neuronal specificity of somatic L1 retrotransposition in neural progenitors is partially due to the transition of a Sox2/HDAC1 repressor complex to a Wnt-mediated T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activator. The transcriptional switch accompanies chromatin remodelling during neuronal differentiation, allowing a transient stimulation of L1 transcription. The activity of L1 retrotransposons during brain development can have an impact on gene expression and neuronal function, thereby increasing brain-specific genetic mosaicism. Further understanding of the molecular mechanisms that regulate L1 expression should provide new insights into the role of L1 retrotransposition during brain development. Here we show that L1 neuronal transcription and retrotransposition in rodents are increased in the absence of methyl-CpG-binding protein 2 (MeCP2), a protein involved in global DNA methylation and human neurodevelopmental diseases. Using neuronal progenitor cells derived from human induced pluripotent stem cells and human tissues, we revealed that patients with Rett syndrome (RTT), carrying MeCP2 mutations, have increased susceptibility for L1 retrotransposition. Our data demonstrate that L1 retrotransposition can be controlled in a tissue-specific manner and that disease-related genetic mutations can influence the frequency of neuronal L1 retrotransposition. Our findings add a new level of complexity to the molecular events that can lead to neurological disorders.

Fig. 1

MeCP2 silences L1 expression. a, Methylation of the L1 5’UTR-Luc reduced its transcriptional activity. b, Reduction of MeCP2 transcripts correlates with increased L1 promoter activity. c, Increased L1 promoter activity in the absence of MeCP2 but not MBD1. d, L1 RNA levels correlate with MeCP2 expression. e, Expression of the MeCP2-VP16 increased the activity of the L1 5’UTR promoter. f and g, Recruitment of MeCP2 on L1 sequences by ChIP in neural stem cells (NSC) or neurons, using 5’UTR primers (f) and two ORF2 regions (g). h, Occupancy of MeCP2 on the L1 promoter requires DNA methylation. Removal of DNA methylation with 5-Azacytidine (5-Aza) reduced MeCP2 association to L1 promoter. ChIP-qPCR shows enrichment over IgG control precipitation. All experiments show experimental triplicates. Error bars in all panels show s.e.m.

Fig. 2

MeCP2 modulates neuronal L1 retrotransposition in vivo. a, EGFP-positive cells, indicating de novo L1 retrotransposition, were found in several regions of the brain. The images were taken from sections that were highly affected by L1 retrotransposition. Bar=30 μm. b, Quantification of brain sections in MeCP2 KO background revealed more EGFP-positive cells compared to WT (n=6 animals for each group). Error bars show s.d. c, Representative images from a 3-dimensional reconstruction of WT and MeCP2 KO brains carrying the L1-EGFP transgene. Single dots (green) represent neurons that supported L1-EGFP retrotransposition. Olfactory bulb is shown in red, striatum in magenta and cerebellum in cyan. R, rostral; C, caudal; D, dorsal and V, ventral.

Fig. 3

Endogenous L1 retrotransposition in mouse neuroepithelial cells. a, Neuroepithelial cells harvested from E11.5 sibling embryos were synchronized and sorted in individual wells followed by qPCR. b, Neuroepithelial cells in the MeCP2 KO background had higher L1 ORF2 DNA content than WT cells (P<0.001). c, L1 5’UTR primers did not reveal a significant increase in copy number in MeCP2 KO background. d, Non-mobile 5S ribosomal genes were used as controls. e, The difference in the amount of L1 ORF2 DNA in fibroblasts from the different genetic backgrounds was smaller than in the neural lineage. All experiments show experimental triplicates (n=192 cells for each primer pair). Error bars in all panels show s.e.m.

Fig. 4

L1 retrotransposition in RTT patients. a, Schematic view of the NPC differentiation from iPSC followed by L1_RE3-EGFP electroporation. b, Representative images of iPSC-derived NPC expressing EGFP after L1 retrotransposition. Bar=30 μm. c, Quantification of the EGFP-positive cells after transfection. d, Primers for ORF2 were used to multiplex with primers for control sequences, such as the 5S ribosomal gene (5S), the satellite alpha (SATA) region, the human endogenous retrovirus H (HERV) sequence and the 5’UTR. The inverse ratio of ORF2/5S represents the amount of L1 ORF2 sequence in each sample (n=5 individuals/group). Similar results were obtained when different primers/probe for ORF2 (ORF2-2) were multiplex/normalized to other control sequences, using two pair of primers (5’UTR-1 or 5’UTR-2). Error bars show s.e.m, and the experiments were performed in triplicate.