RESUMO
BACKGROUND: The internal promoter in L1 5'UTR is critical for autonomous L1 transcription and initiating retrotransposition. Unlike the human genome, which features one contemporarily active subfamily, four subfamilies (A_I, Gf_I and Tf_I/II) have been amplifying in the mouse genome in the last one million years. Moreover, mouse L1 5'UTRs are organized into tandem repeats called monomers, which are separated from ORF1 by a tether domain. In this study, we aim to compare promoter activities across young mouse L1 subfamilies and investigate the contribution of individual monomers and the tether sequence. RESULTS: We observed an inverse relationship between subfamily age and the average number of monomers among evolutionarily young mouse L1 subfamilies. The youngest subgroup (A_I and Tf_I/II) on average carry 3-4 monomers in the 5'UTR. Using a single-vector dual-luciferase reporter assay, we compared promoter activities across six L1 subfamilies (A_I/II, Gf_I and Tf_I/II/III) and established their antisense promoter activities in a mouse embryonic fibroblast cell line and a mouse embryonal carcinoma cell line. Using consensus promoter sequences for three subfamilies (A_I, Gf_I and Tf_I), we dissected the differential roles of individual monomers and the tether domain in L1 promoter activity. We validated that, across multiple subfamilies, the second monomer consistently enhances the overall promoter activity. For individual promoter components, monomer 2 is consistently more active than the corresponding monomer 1 and/or the tether for each subfamily. Importantly, we revealed intricate interactions between monomer 2, monomer 1 and tether domains in a subfamily-specific manner. Furthermore, using three-monomer 5'UTRs, we established a complex nonlinear relationship between the length of the outmost monomer and the overall promoter activity. CONCLUSIONS: The laboratory mouse is an important mammalian model system for human diseases as well as L1 biology. Our study extends previous findings and represents an important step toward a better understanding of the molecular mechanism controlling mouse L1 transcription as well as L1's impact on development and disease.
RESUMO
Long interspersed element type 1 (LINE-1; L1) mobilizes during early embryogenesis, neurogenesis, and germ cell development, accounting for 25% of disease-causing heritable insertions and 98% of somatic insertions in cancer. To better understand the regulation and impact of L1 mobilization in the genome, reliable methods for measuring L1 copy number variation (CNV) are needed. Here we present a comprehensive analysis of a droplet digital PCR (ddPCR) based method for quantifying endogenous mouse L1. We provide experimental evidence that ddPCR assays can be designed to target specific L1 subfamilies using diagnostic single nucleotide polymorphisms (SNPs). The target and off-target L1 subfamilies form distinct droplet clusters, which were experimentally verified using both synthetic gene fragments and endogenous L1 derived plasmid clones. We further provide a roadmap for in silico assay design and evaluation of target specificity, ddPCR testing, and optimization for L1 CNV quantification. The assay can achieve a sensitivity of 5% CNV with 8 technical replicates. With 24 technical replicates, it can detect 2% CNV because of the increased precision. The same approach will serve as a guide for the development of ddPCR based assays for quantifying human L1 copy number and any other high copy genomic target sequences.
