Godfried W. van der Heijden

Asymmetry in Histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote

In mammalian fertilization, the paternal genome is delivered to the secondary oocyte by sperm with protamine compacted DNA, while the maternal genome is arrested in meiotic metaphase II. Thus, at the beginning of fertilization, the two gametic chromatin sets are strikingly different. We elaborate on this contrast by reporting asymmetry for histone H3 type in the pre-S-phase zygote when male chromatin is virtually devoid of histone H3.1/3.2. Localization of the histone H3.3/H4 assembly factor Hira with the paternal chromatin indicates the presence of histone H3.3. In conjunction with this, we performed a systematic immunofluorescence analysis of histone N-tail methylations at position H3K4, H3K9, H3K27 and H4K20 up to the young pronucleus stage and show that asymmetries reported earlier are systematic for virtually all di- and tri-methylations but not for mono-methylation of H3K4 and H4K20, the only marks studied present in the early male pronucleus. For H4K20 the expanding male chromatin is rapidly mono-methylated. This coincides with the formation of maternally derived nucleosomes, a process which is observed as early as sperm chromatin decondensation occurs. Absence of tri-methylated H3K9, tri-methylated H4K20 and presence of loosely anchored HP1-beta combined with the homogenous presence of mono-methylated H4K20 suggests the absence of a division of the paternal chromatin in eu- and heterochromatin. In summary the male, in contrast to female G1 chromatin, is uniform and contains predominantly histone H3.3 as histone H3 variant.

Mouse Maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis

Tight control of transposon activity is essential for the integrity of the germline. Recently, a germ-cell-specific organelle, nuage, was proposed to play a role in transposon repression. To test this hypothesis, we disrupted a murine homolog of a Drosophila nuage protein Maelstrom. Effects on male meiotic chromosome synapsis and derepression of transposable elements (TEs) were observed. In the adult Mael(-/-) testes, LINE-1 (L1) derepression occurred at the onset of meiosis. As a result, Mael(-/-) spermatocytes were flooded with L1 ribonucleoproteins (RNPs) that accumulated in large cytoplasmic enclaves and nuclei. Mael(-/-) spermatocytes with nuclear L1 RNPs exhibited massive DNA damage and severe chromosome asynapsis even in the absence of SPO11-generated meiotic double-strand breaks. This study demonstrates that MAEL, a nuage component, is indispensable for the silencing of TEs and identifies the initiation of meiosis as an important step in TE control in the male germline.

Cytoplasmic Compartmentalization of the Fetal piRNA Pathway in Mice

Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic “cementing material” first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function.

Sperm-derived histones contribute to zygotic chromatin in humans

Backgroundabout 15% to 30% of the DNA in human sperm is packed in nucleosomes and transmission of this fraction to the embryo potentially serves as a mechanism to facilitate paternal epigenetic programs during embryonic development. However, hitherto it has not been established whether these nucleosomes are removed like the protamines or indeed contribute to paternal zygotic chromatin, thereby potentially contributing to the epigenome of the embryo.Resultsto clarify the fate of sperm-derived nucleosomes we have used the deposition characteristics of histone H3 variants from which follows that H3 replication variants present in zygotic paternal chromatin prior to S-phase originate from sperm. We have performed heterologous ICSI by injecting human sperm into mouse oocytes. Probing these zygotes with an antibody highly specific for the H3.1/H3.2 replication variants showed a clear signal in the decondensed human sperm chromatin prior to S-phase. In addition, staining of human multipronuclear zygotes also showed the H3.1/H3.2 replication variants in paternal chromatin prior to DNA replication.Conclusionthese findings reveal that sperm-derived nucleosomal chromatin contributes to paternal zygotic chromatin, potentially serving as a template for replication, when epigenetic information can be copied. Hence, the execution of epigenetic programs originating from transmitted paternal chromatin during subsequent embryonic development is a logical consequence of this observation.

DNA double-strand break repair in parental chromatin of mouse zygotes, the first cell cycle as an origin of de novo mutation

In the human, the contribution of the sexes to the genetic load is dissimilar. Especially for point mutations, expanded simple tandem repeats and structural chromosome mutations, the contribution of the male germline is dominant. Far less is known about the male germ cell stage(s) that are most vulnerable to mutation contraction. For the understanding of de novo mutation induction in the germline, mechanistic insight of DNA repair in the zygote is mandatory. At the onset of embryonic development, the parental chromatin sets occupy one pronucleus (PN) each and DNA repair can be regarded as a maternal trait, depending on proteins and mRNAs provided by the oocyte. Repair of DNA double-strand breaks (DSBs) is executed by non-homologous end joining (NHEJ) and homologous recombination (HR). Differentiated somatic cells often resolve DSBs by NHEJ, whereas embryonic stem cells preferably use HR. We show NHEJ and HR to be both functional during the zygotic cell cycle. NHEJ is already active during replacement of sperm protamines by nucleosomes. The kinetics of G1 repair is influenced by DNA-PK(cs) hypomorphic activity. Both HR and NHEJ are operative in S-phase, HR being more active in the male PN. DNA-PK(cs) deficiency upregulates the HR activity. Both after sperm remodeling and at first mitosis, spontaneous levels of gammaH2AX foci (marker for DSBs) are high. All immunoflurescent indices of DNA damage and DNA repair point at greater spontaneous damage and induced repair activity in paternal chromatin in the zygote.

A role for retrotransposon LINE-1 in fetal oocyte attrition in mice.

Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, an L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition. We demonstrate that experimental elevation of L1 expression correlates with increased MPI defects, FOA, oocyte aneuploidy, and embryonic lethality. Conversely, reverse transcriptase (RT) inhibitor AZT has a profound effect on the FOA dynamics and meiotic recombination, and it implicates an RT-dependent trigger in oocyte elimination in early MPI. We propose that FOA serves to select oocytes with limited L1 activity that are therefore best suited for the next generation.

Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications

The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos.

Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice

Pachytene piRNAs are a class of Piwi‐interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon‐derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor‐domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael129‐null mutant testes possess low levels of piRNAs derived from MAEL‐associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.

Transient relaxation of transposon silencing at the onset of mammalian meiosis.

Postmigratory mouse primordial germ cells (PGCs) undergo extensive epigenetic remodeling that includes DNA methylation (DM) reprogramming of imprinted genes and, surprisingly, of transposable elements (TEs). Given the danger posed by TEs to the integrity of the germline, even a brief derepression of TEs is counterintuitive and puzzling. In the male fetal gonocytes, a sophisticated repressive mechanism that uses DM and TE-targeting piRNAs has evolved to stably silence TEs. A recent study has further increased the complexity of this problem by revealing that TE silencing is alleviated specifically at the onset of meiosis in testes lacking MAEL, a piRNA pathway protein. These observations and prior work of others are consistent with existence of an additional reprogramming event, transient relaxation of transposon silencing (TRTS), at the onset of both male and female meiosis in mice. In this Point of View we propose that TE expression is inherent to mammalian meiosis and discuss potential functional significance of this phenomenon.

Intracellular detection of cytosine incorporation in genomic DNA by using 5-ethynyl-2'-deoxycytidine.

5‐Ethynyl‐2′‐deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA‐centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo− and DNA polymerase β. Incorporation of 5‐ethynyl‐2′‐deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC‐containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.