[Show abstract][Hide abstract]ABSTRACT: Decease in H3K9me2 in Setdb1 KO growing oocytes.
Ovarian sections of 2-month-old control and Setdb1 KO mice were analyzed by immunohistochemistry (IHC) for H3K9me2 (A), H3K9me1, H3K9me3 (B), or H3K4me2 (C), as indicated. Representative staining patterns of growing oocytes are shown, and their nuclei are indicated by arrows. Scale bars, 50 μm.
[Show abstract][Hide abstract]ABSTRACT: Setdb1 deficiency has no effect on oocyte growth.
(A) Periodic acid-Schiff (PAS)-hematoxylin staining showing the histological features of ovaries from 2-month old control and Setdb1 KO mice. CL, corpus luteum. Scale bar, 500 μm. (B) Quantification of follicles from control and Setdb1 KO ovaries. Ovarian sections were examined by microscopy, and follicles of various stages were determined by morphology and counted. The data are presented as the mean ± SEM of 8 ovarian sections from 2 mice for each genotype. (C) Representative bright-field microscope images of control and Setdb1 KO fully-grown GV oocytes showing no difference in morphology. Arrows indicate the prominent nucleoli characteristic of GV oocytes. Scale bar, 50 μm. (D) The numbers of fully-grown GV oocytes harvested from the ovaries of control and Setdb1 KO mice are presented as the mean ± SEM (data from 5 control and 6 Setdb1 KO mice).
[Show abstract][Hide abstract]ABSTRACT: Oocyte-specific deletion of Setdb1.
(A) Schematic diagrams of the Setdb1 alleles. Exons are shown as black bars. Exon 16, flanked by loxP sites (shown as triangles) in the conditional allele, encodes part of the catalytic bifurcated SET domain. The locations of the primers used for genotyping (F1, F2, and R1) are indicated. (B) Mating scheme used to produce Setdb1 knockout (KO) and control mice. (C) Representative PCR genotyping results using tail-tip genomic DNA. For each sample, the left lane (lane 1, 3, 5, or 7) is Cre PCR, and the right lane (lane 2, 4, 6, or 8) is Setdb1 allele PCR.
[Show abstract][Hide abstract]ABSTRACT: Author
During oogenesis, oocytes accumulate transcripts and proteins that support meiotic maturation and early embryogenesis. Although a number of such maternal-effect factors have been identified, our knowledge about the molecular machinery that drives meiotic progression and maternal-to-zygotic transition is still limited. In particular, the functional significance of epigenetic changes, which accompany meiotic maturation and early embryogenesis, and the key epigenetic regulators involved are largely unknown. Here, we identify Setdb1, a lysine methyltransferase specific for the repressive histone H3 lysine 9 (H3K9) methylation, as a maternal-effect factor that is essential for meiotic progression in oocytes and mitotic cell divisions in early embryos in mouse. We show that Setdb1 is highly expressed in growing oocytes and directly represses the expression Cdc14b, a phosphatase that inhibits meiotic progression. Setdb1 is also required to repress retrotransposons and maintain genomic stability in oocytes. Embryos derived from Setdb1-depleted oocytes show severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. The roles of Setdb1 in meiotic progression and preimplantation development require its catalytic activity. Our findings demonstrate that Setdb1 is an important regulator of Cdc14b, thus uncovering a molecular link between the epigenetic machinery and the major signaling pathway that drives meiotic progression.
[Show abstract][Hide abstract]ABSTRACT: Setdb1 binding and H3K9me3 enrichment in meiosis genes in mouse ES cells.
Shown are genome browser screenshots of the Cdc14b, Cdc25b, Bub1b, and Ppp2cb loci showing Setdb1 and H3K9me3 ChIP-Seq data in mouse ES cells (from Bilodeau et al. 2009 ).
[Show abstract][Hide abstract]ABSTRACT: Plasmid constructs used as templates for in vitro transcription for the production of mRNAs encoding Flag-tagged WT Setdb1 or catalytically inactive Setdb1 (C1243A).
Flag-Setdb1 or Flag-C1243A cDNA was inserted into the SpeI-EcoRI sites of pBluescript KS. The constructs were linearized with SalI digestion before being used for in vitro transcription. The location of the C1243A point mutation is indicated.
[Show abstract][Hide abstract]ABSTRACT: MG132 improves GVBD rate of Setdb1 KO oocytes.
Control and Setdb1 KO GV oocytes were collected in M2 medium supplemented with 200 M of IBMX so as to prevent oocytes from undergoing GVBD. Setdb1 KO oocytes were treated with DMSO or MG132 (10 μM) for 4 hours. After washing, control and Setdb1 KO oocytes were cultured in IBMX-free M16 medium for 2 hours. (A) Representative bright-field microscope images of control oocytes and Setdb1 KO oocytes treated with or without MG132. Arrows indicate the prominent nucleoli characteristic of GV oocytes. Scale bars, 50 μm. (B) The percentages of GV, GVBD/MI, and abnormal oocytes. The numbers of oocytes analyzed are indicated.
[Show abstract][Hide abstract]ABSTRACT: Effect of Cdc14b knockdown on spindle phenotype during MI.
GV oocytes were harvested from control and Setdb1 KO mice. Setdb1 KO oocytes were microinjected with either control siRNA or Cdc14b siRNA. The injected oocytes, as well as control GV oocytes, were incubated in IBMX-containing medium for 24 hours to allow siRNA-mediated Cdc14b depletion to occur while maintaining GV arrest and, following IBMX washout, were allowed to mature in vitro for another 6 hours. Oocytes were immunostained for α-tubulin (green) and DNA (blue) to examine spindle and chromosome structures. (A) Representative IF images showing MI oocytes with normal and abnormal spindle structures. (B) Percentages of MI oocytes with spindle defects in the indicated groups. The total number of MI oocytes examined were: 32 control, 30 Setdb1 KO injected with control siRNA, and 34 Setdb1 KO injected with Cdc14b siRNA. Statistical comparisons were made using one-way ANOVA. *P < 0.05; **P < 0.01.
[Show abstract][Hide abstract]ABSTRACT: Mammalian oocytes are arrested at prophase I until puberty when hormonal signals induce the resumption of meiosis I and progression to meiosis II. Meiotic progression is controlled by CDK1 activity and is accompanied by dynamic epigenetic changes. Although the signalling pathways regulating CDK1 activity are well defined, the functional significance of epigenetic changes remains largely unknown. Here we show that LSD1, a lysine demethylase, regulates histone H3 lysine 4 di-methylation (H3K4me2) in mouse oocytes and is essential for meiotic progression. Conditional deletion of Lsd1 in growing oocytes results in precocious resumption of meiosis and spindle and chromosomal abnormalities. Consequently, most Lsd1-null oocytes fail to complete meiosis I and undergo apoptosis. Mechanistically, upregulation of CDC25B, a phosphatase that activates CDK1, is responsible for precocious meiotic resumption and also contributes to subsequent spindle and chromosomal defects. Our findings uncover a functional link between LSD1 and the major signalling pathway governing meiotic progression.
Full-text Article · Dec 2015 · Nature Communications
[Show abstract][Hide abstract]ABSTRACT: Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.
Full-text Article · Nov 2015 · Genes & development
[Show abstract][Hide abstract]ABSTRACT: SETDB1, a histone methyltransferase responsible for methylation of histone H3 lysine 9 (H3K9), is involved in maintenance of embryonic stem (ES) cells and early embryonic development of the mouse. However, how SETDB1 regulates gene expression during development is largely unknown. Here, we characterized genome-wide SETDB1 binding and H3K9 trimethylation (H3K9me3) profiles in mouse ES cells and uncovered two distinct classes of SETDB1 binding sites, termed solo and ensemble peaks. The solo peaks were devoid of H3K9me3 and enriched near developmental regulators while the ensemble peaks were associated with H3K9me3. A subset of the SETDB1 solo peaks, particularly those near neural development related genes, was found to be associated with Polycomb Repressive Complex 2 (PRC2) as well as PRC2-interacting proteins JARID2 and MTF2. Genetic deletion of Setdb1 reduced EZH2 binding as well as histone 3 lysine 27 (H3K27) trimethylation level at SetDB1 solo peaks and facilitated neural differentiation. Furthermore, we found that H3K27me3 inhibits SETDB1 methyltransferase activity. The currently identified reciprocal action between SETDB1 and PRC2 reveals a novel mechanism underlying ES cell pluripotency and differentiation regulation.
Published by Cold Spring Harbor Laboratory Press.
[Show abstract][Hide abstract]ABSTRACT: DNA methylation plays a critical role in the regulation of chromatin structure and gene expression and is involved in a variety of biological processes. The levels and patterns of DNA methylation are regulated by both DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and 'demethylating' proteins, including the ten-eleven translocation (TET) family of dioxygenases (TET1, TET2 and TET3). The effects of DNA methylation on chromatin and gene expression are largely mediated by methylated DNA 'reader' proteins, including MeCP2. Numerous mutations in DNMTs, TETs and MeCP2 have been identified in cancer and developmental disorders, highlighting the importance of the DNA methylation machinery in human development and physiology. In this review, we describe these mutations and discuss how they may lead to disease phenotypes.
[Show abstract][Hide abstract]ABSTRACT: Changes in gene expression and activity underlie the formation of cancerous tumors. Historically, alterations (deletions, point mutations, translocations, etc.) in the DNA genome of the cell were believed to be the basis for tumor formation, but recent studies have demonstrated that epigenetic modifications, which involve the regulated addition of chemical groups to DNA and histones, also profoundly affect gene expression and, thus, cancer. Both genomic region compaction and gene expression are modulated by epigenetic modifications, which are deposited by specific enzymes (known as "writers"), and subsequently recognized by effector proteins ("readers"). Most, if not all, epigenetic marks are reversible, and various enzymes ("erasers") remove these marks. The complex interplay of these three classes of proteins controls gene transcription, and defects in this system contribute to cancer initiation and progression. This chapter will introduce the key concepts surrounding these three types of proteins, with a particular focus on methyl and acetyl marks.
[Show abstract][Hide abstract]ABSTRACT: Cellular differentiation is, by definition, epigenetic. Genome-wide profiling of pluripotent cells and differentiated cells suggests global chromatin remodelling during differentiation, which results in a progressive transition from a fairly open chromatin configuration to a more compact state. Genetic studies in mouse models show major roles for a variety of histone modifiers and chromatin remodellers in key developmental transitions, such as the segregation of embryonic and extra-embryonic lineages in blastocyst stage embryos, the formation of the three germ layers during gastrulation and the differentiation of adult stem cells. Furthermore, rather than merely stabilizing the gene expression changes that are driven by developmental transcription factors, there is emerging evidence that chromatin regulators have multifaceted roles in cell fate decisions.
[Show abstract][Hide abstract]ABSTRACT: Somatic heterozygous mutations of the DNA methyltransferase gene DNMT3A occur frequently in acute myeloid leukemia and other hematological malignancies, with the majority (~60%) of mutations affecting a single amino acid, Arg882 (R882), in the catalytic domain. While the mutations impair DNMT3A catalytic activity in vitro, their effects on DNA methylation in cells have not been explored. Here, we show that exogenously expressed mouse Dnmt3a proteins harboring the corresponding R878 mutations largely fail to mediate DNA methylation in murine embryonic stem (ES) cells, but are capable of interacting with wild-type Dnmt3a and Dnmt3b. Co-expression of the Dnmt3a R878H (histidine) mutant protein results in inhibition of the ability of wild-type Dnmt3a and Dnmt3b to methylate DNA in murine ES cells. Furthermore, expression of Dnmt3a R878H in ES cells containing endogenous Dnmt3a or Dnmt3b induces hypomethylation. These results suggest that the DNMT3A R882 mutations, in addition to being hypomorphic, have dominant-negative effects.
[Show abstract][Hide abstract]ABSTRACT: Methylation of cytosines is a major epigenetic modification in mammalian genomes. The levels and patterns of DNA methylation are the results of the opposing actions of methylating and demethylating machineries. Over the past two decades, great progress has been made in elucidating the methylating machinery including the identification and functional characterization of the DNA methyltransferases (Dnmts). However, the mechanisms of demethylation and the major players involved had been elusive. A major breakthrough came in 2009, when the ten-eleven translocation (Tet) family of proteins was discovered as 5-methylcytosine (5mC) dioxygenases that convert 5mC to 5-hydroxymethylcytosine (5hmC). Studies in the past several years have established that 5hmC serves as an intermediate in DNA demethylation and that Tet proteins have important roles in epigenetic reprogramming in early embryos and primordial germ cells. In this review, we discuss recent advances in this exciting field, focusing on the role of Tet proteins in mammalian development.Journal of Human Genetics advance online publication, 30 May 2013; doi:10.1038/jhg.2013.63.
[Show abstract][Hide abstract]ABSTRACT: Modeling of the location of the point mutations in the 3D structure of Lsd1. Computer modeling of the structure of Lsd1 indicates that the point mutation at position 413 is present at the base of the tower domain, and may have effects on the structure of both the tower and the amine oxidase domain. The mutation at position 448 occurs at a residue that is known to be involved in binding to CoREST, and as such may affect Lsd1 protein-protein interactions.
[Show abstract][Hide abstract]ABSTRACT: Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic Lsd1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Molecular analyses revealed hyperphosphorylation of E-cadherin in the hearts of mutant animals. These results identify a previously unknown role for Lsd1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.