Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition

Revealing the mechanisms for neuronal somatic diversification remains a central challenge for understanding individual differences in brain organization and function. Here we show that an engineered human LINE-1 (for long interspersed nuclear element-1; also known as L1) element can retrotranspose in neuronal precursors derived from rat hippocampus neural stem cells. The resulting retrotransposition events can alter the expression of neuronal genes, which, in turn, can influence neuronal cell fate in vitro. We further show that retrotransposition of a human L1 in transgenic mice results in neuronal somatic mosaicism. The molecular mechanism of action is probably mediated through Sox2, because a decrease in Sox2 expression during the early stages of neuronal differentiation is correlated with increases in both L1 transcription and retrotransposition. Our data therefore indicate that neuronal genomes might not be static, but some might be mosaic because of de novo L1 retrotransposition events.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62714/1/nature03663.pd

Co-chaperones are limiting in a depleted chaperone network

To probe the limiting nodes in the chaperoning network which maintains cellular proteostasis, we expressed a dominant negative mutant of heat shock factor 1 (dnHSF1), the regulator of the cytoplasmic proteotoxic stress response. Microarray analysis of non-stressed dnHSF1 cells showed a two- or more fold decrease in the transcript level of 10 genes, amongst which are the (co-)chaperone genes HSP90AA1, HSPA6, DNAJB1 and HSPB1. Glucocorticoid signaling, which requires the Hsp70 and the Hsp90 folding machines, was severely impaired by dnHSF1, but fully rescued by expression of DNAJA1 or DNAJB1, and partially by ST13. Expression of DNAJB6, DNAJB8, HSPA1A, HSPB1, HSPB8, or STIP1 had no effect while HSP90AA1 even inhibited. PTGES3 (p23) inhibited only in control cells. Our results suggest that the DNAJ co-chaperones in particular become limiting in a depleted chaperoning network. Our results also suggest a difference between the transcriptomes of cells lacking HSF1 and cells expressing dnHSF1

Many LINE1 elements contribute to the transcriptome of human somatic cells

Over 600 LINE 1 elements are shown to be transcribed in humans; 400 of these are full-length elements in the reference genome

Intronic L1 Retrotransposons and Nested Genes Cause Transcriptional Interference by Inducing Intron Retention, Exonization and Cryptic Polyadenylation

Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown.Using in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3' ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs) and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals.Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression

Imaging Drosophila brain by combining cryo soft X ray microscopy of thick vitreous sections and cryo electron microscopy of ultrathin vitreous sections

Cryo soft X ray microscopy is an emerging imaging tool complementary to cryo electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower resolution. We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo soft X ray microscopy of thick vitreous sections, and cryo electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 m, by tuning the sectioning speed of the cryo microtome. A fluorescent stereo microscope mounted on the cryo microtome allowed us to target the regions of interest after GFP labeling of synapses. Thick cryo sections were imaged by cryo soft X ray microscopy at a resolution better than 25 nm, while ultrathin cryo sections of the same regions were explored in parallel at the nanometre level of resolution by cryo electron microscop

Aberrant base excision repair pathway of oxidatively damaged DNA: Implications for degenerative diseases

Abstract In cellular organisms composition of DNA is constrained to only four nucleobases A, G, T and C, except for minor DNA base modifications such as methylation which serves for defence against foreign DNA or gene expression regulation. Interestingly, this severe evolutionary constraint among other things demands DNA repair systems to discriminate between regular and modified bases. DNA glycosylases specifically recognize and excise damaged bases among vast majority of regular bases in the base excision repair (BER) pathway. However, the mismatched base pairs in DNA can occur from a spontaneous conversion of 5-methylcytosine to thymine and DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved special DNA repair systems that target the non-damaged DNA strand in a duplex to remove mismatched regular DNA bases. Mismatch-specific adenine- and thymine-DNA glycosylases (MutY/MUTYH and TDG/MBD4, respectively) initiated BER and mismatch repair (MMR) pathways can recognize and remove normal DNA bases in mismatched DNA duplexes. Importantly, in DNA repair deficient cells bacterial MutY, human TDG and mammalian MMR can act in the aberrant manner: MutY and TDG removes adenine and thymine opposite misincorporated 8-oxoguanine and damaged adenine, respectively, whereas MMR removes thymine opposite to O6-methylguanine. These unusual activities lead either to mutations or futile DNA repair, thus indicating that the DNA repair pathways which target non-damaged DNA strand can act in aberrant manner and introduce genome instability in the presence of unrepaired DNA lesions. Evidences accumulated showing that in addition to the accumulation of oxidatively damaged DNA in cells, the aberrant DNA repair can also contribute to cancer, brain disorders and premature senescence. For example, the aberrant BER and MMR pathways for oxidized guanine residues can lead to trinucleotide expansion that underlies Huntington's disease, a severe hereditary neurodegenerative syndrome. This review summarises the present knowledge about the aberrant DNA repair pathways for oxidized base modifications and their possible role in age-related diseases