UBC Theses and Dissertations
Transcriptional repression of retrotransposons in mouse germline Liu, Sheng
Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at approximately embryonic day 15.5 (E15.5) in prospermatogonia. However earlier in germline development, the genome, including most retrotransposons, is progressively demethylated. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, raising the question whether repressive histone methylations play a role in silencing of retrotransposons at this stage of development. To answer this question, I first focused on developing low input assays for profiling histone modifications, DNA methylation and transcription from rare cell populations. In close collaboration with Dr. Julie Brind’Amour, I was able to develop the “SmallCell” protocol package, which enables chromatin immunoprecipitation, bisulfite conversion of DNA, RNA isolation-reverse transcription using ~1000 cells per assay, but also construction of sequencing library from pictograms of DNA. This allows profiling of epigenetic information at both locus-specific and genome-wide scales. I then developed the “InterSeq” software (R package) to intersect and explore different types of epigenomic data. This package allows converting sequencing data into genomic interval measures in spreadsheet (SeqData), interfacing this spreadsheet into flowcytometry data (SeqFrame), and an intuitive graphical interface to gate and explore the inter-relationship between different types of epigenomic sequencing data similar to flowcytometry (SeqViz). With these tools we first determined whether retrotransposons are marked by H3K9me3 and H3K27me3. Although these repressive histone modifications are found predominantly on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated long terminal repeats and LINE1 elements. Germline-specific conditional knockout of the H3K9 methyltransferase SETDB1 yields a decrease of both marks and DNA methylation at H3K9me3-enriched retrotransposon families. Strikingly, Setdb1 knockout E13.5 PGCs show concomitant derepression of many marked ERVs, including IAP, ETn, and ERVK10C elements, and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male E13.5 PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline.
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