Biology subjects

Sanchez-Luque, F. J.

Publications and source records attributed to Sanchez-Luque, F. J..

7 recordsLinked to original sources

bioRxiv2023

Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation

Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. Here we show that vitC increases the expression of evolutionarily young LINE-1 (L1) elements in mouse ESCs. We find that TET activity is dispensable for these effects, and that instead L1 upregulation occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, which could impact the genetic and epigenetic stability of human pluripotent stem cells.

genetics↗

bioRxiv2022

Mouse L1s fade with age: a methylation-enforced mechanism for attenuation of L1 retrotransposition potential

Mice harbor [~]2,800 intact copies of the retrotransposon Long Interspersed Element 1 (L1). The in vivo retrotransposition capacity of an L1 copy is defined by both its sequence integrity and epigenetic status, including DNA methylation of the monomeric units constituting young mouse L1 promoters. Locus-specific L1 methylation dynamics during development may therefore elucidate and explain spatiotemporal niches of endogenous retrotransposition, but remain unresolved. Here, we interrogate the retrotransposition efficiency and epigenetic fate of source (donor) L1s, identified as mobile in vivo. We demonstrate that promoter monomer loss consistently attenuates the relative retrotransposition potential of their offspring (daughter) L1 insertions. We also observe that most donor/daughter L1 pairs are efficiently methylated upon differentiation in vivo and in vitro. We employ Oxford Nanopore Technologies (ONT) long-read sequencing to resolve L1 methylation genome-wide and with locus-specific resolution, revealing a distinctive "smile" pattern in methylation levels across the L1 promoter region and thereby elucidating a molecular mechanism potentially underpinning L1 promoter shortening. Together, our results offer a novel perspective on the interplay between epigenetic repression, L1 evolution, and genome stability.

genetics↗

bioRxiv2022

LINE-1 retrotransposon activation intrinsic to interneuron development

Retrotransposons are a reservoir of cis-regulatory innovation1-3. Developmental programs that activate these elements could, in principle, manifest in lineage-specific retrotransposition. Somatic LINE-1 (L1) retrotransposon insertions have been detected in human and non-human primate neurons4-7. It is however unknown whether L1 is mobile in only some neuronal lineages, or therein regulates neurodevelopmental genes. Here, we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development8-10. PV+ neurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters, and express full-length L1 mRNAs and proteins. Via nanopore long-read sequencing, we identify unmethylated L1 promoters proximal to PV+ neuron genes. One such L1, which promotes transcription of a novel CAPS2 gene isoform, significantly enhances neuron morphological complexity when phenotyped in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV+ neuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

genomics↗

bioRxiv2022

Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements (TEs) and prevent TE-mediated mutagenesis. Here we applied short-read or Oxford Nanopore Technologies (ONT) long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs were rare, we found 83 de novo TE insertions, including examples intronic to Brca1 and Dmd. LINE-1 (L1) retrotransposons were profoundly hypomethylated in miPSCs, beyond other TEs and the genome overall, and harbored alternative protein-coding gene promoters. Treatment with the L1 inhibitor lamivudine did not hinder reprogramming and efficiently blocked endogenous retrotransposition, as detected by ONT sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.

genomics↗

bioRxiv2021

Human genome integration of SARS-CoV-2 contradicted by long-read sequencing

A recent study proposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we applied deep (>50x) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2, and did not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolved 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV) positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions via ONT sequencing. That we found no evidence of SARS-CoV-2 integration suggests such events are, at most, extremely rare in vivo, and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.

genomics↗

bioRxiv2020

Nanopore sequencing enables comprehensive transposable element epigenomic profiling

We apply long-read nanopore sequencing and a new tool, TLDR (Transposons from Long Dirty Reads), to directly infer CpG methylation of new and extant human transposable element (TE) insertions in hippocampus, heart, and liver, as well as paired tumour and non-tumour liver. Whole genome TLDR analysis greatly facilitates studies of TE biology as complete insertion sequences and their epigenetic modifications are readily obtainable.

genomics↗

bioRxiv2019

LINE-1 retrotransposition impacts the genome of human pre implantation embryos and extraembryonic tissues

Long Interspersed Element 1 (LINE-1/L1) is an abundant retrotransposon that has greatly impacted human genome evolution. LINE-1s are responsible for the generation of millions of insertions in the current human population. The characterization of sporadic cases of mosaic individuals carrying pathogenic L1-insertions, suggest that heritable insertions occurs during early embryogenesis. However, the timing and potential genomic impact of LINE-1 mobilization during early embryogenesis is unknown. Here, we demonstrate that inner cell mass of human pre-implantation embryos support the expression and retrotransposition of LINE -1s. Additionally, we show that LINE-1s are expressed in trophectoderm cells of embryos, and identify placenta-restricted endogenous LINE-1 insertions in newborns. Using human embryonic stem cells as a model of post-implantation epiblast cells, we demonstrate ongoing LINE-1 retrotransposition, which can impact expression of targeted genes. Our data demonstrate that LINE-1 retrotransposition starts very shortly after fertilization and may represent a previously underappreciated factor in human biology and disease.

genomics↗