Microarray analysis of LTR retrotransposon silencing identifies Hdac1 as a regulator of retrotransposon expression in mouse embryonic stem cells

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells

Roles of the pluripotency associated Tex19.1 gene in mouse embryonic and germline development

Chromosome segregation errors that occur in the developing germline generate aneuploidies which are among the leading causes of embryonic lethality, spontaneous abortions and chromosomal disorders, such as Down’s syndrome. Compared to other species, human oocytes appear to be particularly prone to suffer chromosome missegregation and the risk of aneuploid pregnancies in humans increases drastically with maternal age. Despite its particular importance for human health, relatively little is known about the basis for the high incidence of aneuploidies in human oocytes and the maternal-age effect. The identification and analysis of molecular pathways that promote genetic and chromosomal stability is important for our understanding of mechanisms that lead to aneuploidy and how it can be prevented. Here, I examine the role of the pluripotency associated Tex19.1 gene, in preventing aneuploidy during mouse female germ cell development. I demonstrate that Tex19.1-/- females are subfertile when mated with wild type males due to defects in chromosome segregation during meiosis. In contrast to Tex19.1-/- male gem cells, synaptonemal complex formation appears to be completed normally in Tex19.1-/- females but high levels of aneuploidy are evident during the second meiotic stages of oogenesis. The Tex19.1-/- females transmit these aneuploidies to their offspring likely resulting in the observed embryonic death and subfertility. In addition to its role in the female germline, I investigated the function of Tex19.1 during embryonic development. I found that Tex19.1-/- knockout mice are born at a sub- Mendelian frequency and this reduction is exacerbated in diapaused embryos, suggesting that Tex19.1 plays a role during a stage where a pluripotent state is maintained for a prolonged period of time. Furthermore, I identified high levels of aneuploidy accumulating in pluripotent stem cells in the absence of Tex19.1

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Latent regulatory potential of human-specific repetitive elements

At least half of the human genome is derived from repetitive elements, which are often lineage specific and silenced by a variety of genetic and epigenetic mechanisms. Using a transchromosomic mouse strain that transmits an almost complete single copy of human chromosome 21 via the female germline, we show that a heterologous regulatory environment can transcriptionally activate transposon-derived human regulatory regions. In the mouse nucleus, hundreds of locations on human chromosome 21 newly associate with activating histone modifications in both somatic and germline tissues, and influence the gene expression of nearby transcripts. These regions are enriched with primate and human lineage-specific transposable elements, and their activation corresponds to changes in DNA methylation at CpG dinucleotides. This study reveals the latent regulatory potential of the repetitive human genome and illustrates the species specificity of mechanisms that control it

Systematic Identification of Factors for Provirus Silencing in Embryonic Stem Cells

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome

Dissecting the meiotic defects of Tex19.1-/- mouse spermatocytes

The maintenance of genomic stability through suppression of retrotransposon activity is vital for the avoidance of potentially mutagenic genomic disruption caused by retrotransposition. Germline development is a particularly important phase for retrotransposon silencing as retrotransposition events here have the potential for transmission to the entire embryo, threatening the health of offspring. A collection of germline genome defence genes are required for the suppression of retrotransposons in the developing germline of male mice (e.g. Tex19.1, Dazl, Mili, Miwi2, Gasz, Mov10l1, Mael, Dnmt3l), all of which trigger meiotic prophase arrest when mutated. I have analysed the meiotic defects which arise in Tex19.1-/- male mice to contribute to the understanding of the fundamental mechanisms required for successful completion of meiosis and to investigate the involvement of retrotransposon silencing in this process. The absence of TEX19.1 in male mice causes infertility; with failed chromosome synapsis in ~50% of pachytene nuclei and associated apoptosis, as well as individual univalent chromosomes in 67% of remaining nuclei progressing to metaphase I. Where studied, failed chromosome synapsis is a common feature of germline genome defence mutant spermatocytes. One aim of my studies has been to better understand the mechanism responsible for this failed chromosome synapsis. I have demonstrated that unlike Mael-/- spermatocytes, additional SPO11-independent DNA damage potentially attributable to retrotransposition is not detectable in Tex19.1-/- spermatocytes. Rather, the formation of meiotic DNA double strand breaks (DSBs) is dramatically reduced in early prophase to around 50%, resulting in a reduction in nuclear γH2AX signal, production of SPO11- oligonucleotide complexes and foci formation by early recombination proteins RPA, DMC1 and RAD51. Despite this early reduction, DSB frequency recovers to more normal levels shortly after in zygotene. I have shown that defective pairing of homologous chromosomes by meiotic recombination is likely responsible for the asynapsis previously reported. The initial reduction in DSB frequency could be sufficient to cause failed chromosome synapsis in this mutant, assuming that late-forming DSBs cannot participate effectively in promoting homologous pairing. Alternative hypotheses include altered positioning of DSBs in response to altered chromatin organisation relating to retrotransposon upregulation, misguiding the pairing of homologous chromosomes. Such a model of disruption could also extend to other germline genome defence mutants. I have demonstrated that despite successful pairing of homologous chromosomes in a sub-population of Tex19.1-/- spermatocytes, subsequent progression of these cells through pachytene is delayed. Numerous diverse features of progression are all delayed, including recombination, ubiquitination on autosomes and sex chromosomes, expression of the mid-pachytene marker H1t, and chromosome organisation. The delay identified is related to recombination therefore this feature is likely to stem from the initial defect in DSB formation early in prophase. While some delayed features are probably directly related to recombination, others are not. The coordinated delay observed may suggest the presence of a recombination-sensitive cell-cycle checkpoint operating to regulate progression through pachytene. My research has also aimed to establish the cause of elevated univalent chromosomes not connected by chiasmata in metaphase I Tex19.1-/- spermatocytes. I have demonstrated that that absence of chiasmata is not due to failed crossover formation between synapsed chromosomes. Rather, the frequent observation of individual unsynapsed chromosomes during crossover formation suggests that some spermatocytes with low-level asynapsis are leaking through meiotic checkpoints and are unable to form a crossover before reaching metaphase. Therefore, again this later meiotic defect appears to stem from the initial defect in meiotic DSB formation, the consequences of which vary widely in severity. Remarkably the unsynapsed chromosomes present during crossover formation include both sex chromosomes, and autosomes. Tolerance of an unsynapsed autosome from pachytene into metaphase is an unusual observation in mice and this observation may aid the understanding of spermato cyte quality control mechanisms during this progression. Together these findings have greatly advanced the understanding of the infertility incurred during meiosis in Tex19.1-/- male mice. These findings may also extend to benefit the understanding of other germline genome defence mutants. Diverse observations made during my investigations also reveal a potential system of coordinated progression through pachytene relating to meiotic recombination. The variable severity of the synapsis defects incurred in this mutant appears to have variable effects on spermatocyte survival and could also inform the understanding of meiotic checkpoint sensitivity

Expression Of Line-1 In Human Somatic Tissues And The Factors Correlated With Line- 1 Expression

Despite the long-held assumption that transposons are normally only expressed in the germline, recently we discovered that full length or partial transcripts of LINE1 are frequently found in the somatic cells. However, the extent of variation in LINE1 levels across different tissues and different individuals, and the genes and pathways that are co-expressed with LINE1 are unknown. Co-expressed genes may be candidate genes that are functioning in transposon silencing. Here, we report the extent of variation in L1HS expression levels across cancer tissues and healthy tissues collected for The Cancer Genome Atlas (TCGA). L1HS is overexpressed in most of the cancer types we have studied. Our results confirm earlier reports of higher L1HS expression in the esophagus and stomach tissues. We also show that mitochondrial genes are enriched among the genes whose expression is negatively correlated with L1HS expression and that PHD fingers, bromodomains and KRAB-zinc fingers (KRAB-ZFPs) are enriched among the genes positively co-expressed with L1HS. Additionally, we studied the association of L1HS transcript level with miRNA expression, and we found several candidate miRNAs that are significantly correlated with L1HS expressio

Distinct contributions of DNA methylation and histone acetylation to the genomic occupancy of transcription factors

Epigenetic modifications on chromatin play important roles in regulating gene expression. Although chromatin states are often governed by multilayered structure, how individual pathways contribute to gene expression remains poorly understood. For example, DNA methylation is known to regulate transcription factor binding but also to recruit methyl-CpG binding proteins that affect chromatin structure through the activity of histone deacetylase complexes (HDACs). Both of these mechanisms can potentially affect gene expression, but the importance of each, and whether these activities are integrated to achieve appropriate gene regulation, remains largely unknown. To address this important question, we measured gene expression, chromatin accessibility, and transcription factor occupancy in wild-type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. We observe widespread increases in chromatin accessibility at retrotransposons when HDACs are inhibited, and this is magnified when cells also lack DNA methylation. A subset of these elements has elevated binding of the YY1 and GABPA transcription factors and increased expression. The pronounced additive effect of HDAC inhibition in DNA methylation-deficient cells demonstrates that DNA methylation and histone deacetylation act largely independently to suppress transcription factor binding and gene expression