Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency.

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

The DOT1L-MLLT10 complex regulates male fertility and promotes histone removal during spermiogenesis.

Histone modifications regulate chromatin remodeling and gene expression in development and diseases. DOT1L, the sole histone H3K79 methyltransferase, is essential for embryonic development. Here, we report that DOT1L regulates male fertility in mouse. DOT1L associates with MLLT10 in testis. DOT1L and MLLT10 localize to the sex chromatin in meiotic and post-meiotic germ cells in an inter-dependent manner. Loss of either DOT1L or MLLT10 leads to reduced testis weight, decreased sperm count and male subfertility. H3K79me2 is abundant in elongating spermatids, which undergo the dramatic histone-to-protamine transition. Both DOT1L and MLLT10 are essential for H3K79me2 modification in germ cells. Strikingly, histones are substantially retained in epididymal sperm from either DOT1L- or MLLT10-deficient mice. These results demonstrate that H3K79 methylation promotes histone replacement during spermiogenesis.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

The MOV10 RNA helicase is a dosage-dependent host restriction factor for LINE1 retrotransposition in mice.

Transposable elements constitute nearly half of the mammalian genome and play important roles in genome evolution. While a multitude of both transcriptional and post-transcriptional mechanisms exist to silence transposable elements, control of transposition in vivo remains poorly understood. MOV10, an RNA helicase, is an inhibitor of mobilization of retrotransposons and retroviruses in cell culture assays. Here we report that MOV10 restricts LINE1 retrotransposition in mice. Although MOV10 is broadly expressed, its loss causes only incomplete penetrance of embryonic lethality, and the surviving MOV10-deficient mice are healthy and fertile. Biochemically, MOV10 forms a complex with UPF1, a key component of the nonsense-mediated mRNA decay pathway, and primarily binds to the 3' UTR of somatically expressed transcripts in testis. Consequently, loss of MOV10 results in an altered transcriptome in testis. Analyses using a LINE1 reporter transgene reveal that loss of MOV10 leads to increased LINE1 retrotransposition in somatic and reproductive tissues from both embryos and adult mice. Moreover, the degree of LINE1 retrotransposition inhibition is dependent on the Mov10 gene dosage. Furthermore, MOV10 deficiency reduces reproductive fitness over successive generations. Our findings demonstrate that MOV10 attenuates LINE1 retrotransposition in a dosage-dependent manner in mice.

The N-terminal modification of HORMAD2 causes its ectopic persistence on synapsed chromosomes without meiotic blockade.

In brief: The dissociation of HORMA domain protein 2 (HORMAD2) from the synaptonemal complex is tightly regulated. This study reveals that the N-terminal region of HORMAD2 is critical for its dissociation from synapsed meiotic chromosomes. Abstract: During meiosis, homologous chromosomes undergo synapsis and recombination. HORMA domain proteins regulate key processes in meiosis. Mammalian HORMAD1 and HORMAD2 localize to unsynapsed chromosome axes but are removed upon synapsis by the TRIP13 AAA+ ATPase. TRIP13 engages the N-terminal region of HORMA domain proteins to induce an open conformation, resulting in the disassembly of protein complexes. Here, we report introduction of a 3×FLAG-HA tag to the N-terminus of HORMAD2 in mice. Coimmunoprecipitation coupled with mass spectrometry identified HORMAD1 and SYCP2 as HORMAD2-associated proteins in the testis. Unexpectedly, the N-terminal tagging of HORMAD2 resulted in its abnormal persistence along synapsed regions in pachynema and ectopic localization to telomeres in diplonema. Super-resolution microscopy revealed that 3×FLAG-HA-HORMAD2 was distributed along the central region of the synaptonemal complex, whereas wild-type HORMAD1 persisted along the lateral elements in 3×FLAG-HA-HORMAD2 meiocytes. Although homozygous mice completed meiosis and were fertile, homozygous males exhibited a significant reduction in sperm count. Collectively, these results suggest that the N-terminus of HORMAD2 is important for its timely removal from meiotic chromosome axes.

Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium.

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.

SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination.

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.

Genetic characterization of a missense mutation in the X-linked TAF7L gene identified in an oligozoospermic man†.

Although hundreds of knockout mice show infertility as a major phenotype, the causative genic mutations of male infertility in humans remain rather limited. Here, we report the identification of a missense mutation (D136G) in the X-linked TAF7L gene as a potential cause of oligozoospermia in men. The human aspartate (D136) is evolutionally conserved across species, and its change to glycine (G) is predicted to be detrimental. Genetic complementation experiments in budding yeast demonstrate that the conserved aspartate or its analogous asparagine (N) residue in yeast TAF7 is essential for cell viability and thus its mutation to G is lethal. Although the corresponding D144G substitution in the mouse Taf7l gene does not affect male fertility, RNA-seq analyses reveal alterations in transcriptomic profiles in the Taf7l (D144G) mutant testes. These results support TAF7L mutation as a risk factor for oligozoospermia in humans.

The ssDNA-binding protein MEIOB acts as a dosage-sensitive regulator of meiotic recombination.

Meiotic recombination enables reciprocal exchange of genetic information between parental chromosomes and is essential for fertility. MEIOB, a meiosis-specific ssDNA-binding protein, regulates early meiotic recombination. Here we report that the human infertility-associated missense mutation (N64I) in MEIOB causes protein degradation and reduced crossover formation in mouse testes. Although the MEIOB N64I substitution is associated with human infertility, the point mutant mice are fertile despite meiotic defects. Meiob mutagenesis identifies serine 67 as a critical residue for MEIOB. Biochemically, these two mutations (N64I and S67 deletion) cause self-aggregation of MEIOB and sharply reduced protein half-life. Molecular genetic analyses of both point mutants reveal an important role for MEIOB in crossover formation in late meiotic recombination. Furthermore, we find that the MEIOB protein levels directly correlate with the severity of meiotic defects. Our results demonstrate that MEIOB regulates meiotic recombination in a dosage-dependent manner.

Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development.

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3' UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3' UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3' end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.

A cell-based high-content screen identifies isocotoin as a small molecule inhibitor of the meiosis-specific MEIOB-SPATA22 complex†.

MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB-SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB-SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein-protein interactions or promote degradation of meiosis-specific proteins.

Arginyltransferase ATE1 is targeted to the neuronal growth cones and regulates neurite outgrowth during brain development.

Arginylation is an emerging protein modification mediated by arginyltransferase ATE1, shown to regulate embryogenesis and actin cytoskeleton, however its functions in different physiological systems are not well understood. Here we analyzed the role of ATE1 in brain development and neuronal growth by producing a conditional mouse knockout with Ate1 deletion in the nervous system driven by Nestin promoter (Nes-Ate1 mice). These mice were weaker than wild type, resulting in low postnatal survival rates, and had abnormalities in the brain that suggested defects in neuronal migration. Cultured Ate1 knockout neurons showed a reduction in the neurite outgrowth and the levels of doublecortin and F-actin in the growth cones. In wild type, ATE1 prominently localized to the growth cones, in addition to the cell bodies. Examination of the Ate1 mRNA sequence reveals the existence of putative zipcode-binding sequences involved in mRNA targeting to the cell periphery and local translation at the growth cones. Fluorescence in situ hybridization showed that Ate1 mRNA localized to the tips of the growth cones, likely due to zipcode-mediated targeting, and this localization coincided with spots of localization of arginylated ß-actin, which disappeared in the presence of protein synthesis inhibitors. We propose that zipcode-mediated co-targeting of Ate1 and ß-actin mRNA leads to localized co-translational arginylation of ß-actin that drives the growth cone migration and neurite outgrowth.

The carboxy-terminus of p63 links cell cycle control and the proliferative potential of epidermal progenitor cells.

The transcription factor p63 (Trp63) plays a key role in homeostasis and regeneration of the skin. The p63 gene is transcribed from dual promoters, generating TAp63 isoforms with growth suppressive functions and dominant-negative ΔNp63 isoforms with opposing properties. p63 also encodes multiple carboxy (C)-terminal variants. Although mutations of C-terminal variants have been linked to the pathogenesis of p63-associated ectodermal disorders, the physiological role of the p63 C-terminus is poorly understood. We report here that deletion of the p63 C-terminus in mice leads to ectodermal malformation and hypoplasia, accompanied by a reduced proliferative capacity of epidermal progenitor cells. Notably, unlike the p63-null condition, we find that p63 C-terminus deficiency promotes expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (Cdkn1a), a factor associated with reduced proliferative capacity of both hematopoietic and neuronal stem cells. These data suggest that the p63 C-terminus plays a key role in the cell cycle progression required to maintain the proliferative potential of stem cells of many different lineages. Mechanistically, we show that loss of Cα, the predominant C-terminal p63 variant in epithelia, promotes the transcriptional activity of TAp63 and also impairs the dominant-negative activity of ΔNp63, thereby controlling p21(Waf1/Cip1) expression. We propose that the p63 C-terminus links cell cycle control and the proliferative potential of epidermal progenitor cells via mechanisms that equilibrate TAp63 and ΔNp63 isoform function.

Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress.

Epigenetic signatures in germ cells, capable of both responding to the parental environment and shaping offspring neurodevelopment, are uniquely positioned to mediate transgenerational outcomes. However, molecular mechanisms by which these marks may communicate experience-dependent information across generations are currently unknown. In our model of chronic paternal stress, we previously identified nine microRNAs (miRs) that were increased in the sperm of stressed sires and associated with reduced hypothalamic-pituitary-adrenal (HPA) stress axis reactivity in offspring. In the current study, we rigorously examine the hypothesis that these sperm miRs function postfertilization to alter offspring stress responsivity and, using zygote microinjection of the nine specific miRs, demonstrated a remarkable recapitulation of the offspring stress dysregulation phenotype. Further, we associated long-term reprogramming of the hypothalamic transcriptome with HPA axis dysfunction, noting a marked decreased in the expression of extracellular matrix and collagen gene sets that may reflect an underlying change in blood-brain barrier permeability. We conclude by investigating the developmental impact of sperm miRs in early zygotes with single-cell amplification technology, identifying the targeted degradation of stored maternal mRNA transcripts including sirtuin 1 and ubiquitin protein ligase E3a, two genes with established function in chromatin remodeling, and this potent regulatory function of miRs postfertilization likely initiates a cascade of molecular events that eventually alters stress reactivity. Overall, these findings demonstrate a clear mechanistic role for sperm miRs in the transgenerational transmission of paternal lifetime experiences.

Polycomb protein SCML2 associates with USP7 and counteracts histone H2A ubiquitination in the XY chromatin during male meiosis.

Polycomb group proteins mediate transcriptional silencing in diverse developmental processes. Sex chromosomes undergo chromosome-wide transcription silencing during male meiosis. Here we report that mouse SCML2 (Sex comb on midleg-like 2), an X chromosome-encoded polycomb protein, is specifically expressed in germ cells, including spermatogonia, spermatocytes, and round spermatids. SCML2 associates with phosphorylated H2AX and localizes to the XY body in spermatocytes. Loss of SCML2 in mice causes defective spermatogenesis, resulting in sharply reduced sperm production. SCML2 interacts with and recruits a deubiquitinase, USP7, to the XY body in spermatocytes. In the absence of SCML2, USP7 fails to accumulate on the XY body, whereas H2A monoubiquitination is dramatically augmented in the XY chromatin. Our results demonstrate that the SCML2/USP7 complex constitutes a novel molecular pathway in modulating the epigenetic state of sex chromosomes during male meiosis.

Accelerated reproductive aging in females lacking a novel centromere protein SYCP2L.

Menopause results from loss of ovarian function and marks the end of a woman's reproductive life. Alleles of the human SYCP2L locus are associated with age at natural menopause (ANM). SYCP2L is a paralogue of the synaptonemal complex protein SYCP2 and is expressed exclusively in oocytes. Here we report that SYCP2L localizes to centromeres of dictyate stage oocytes, which represent the limited pool of primordial oocytes that are formed perinatally and remain arrested till ovulation. Centromere localization of SYCP2L requires its C-terminal portion, which is missing in truncated variants resulting from low-frequency nonsense mutations identified in humans. Female mice lacking SYCP2L undergo a significantly higher progressive loss of oocytes with age compared with wild-type females and are less fertile. Specifically, the pool of primordial oocytes becomes more rapidly depleted in SYCP2L-deficient than in wild-type females, such that with aging, fewer oocytes undergo maturation in developing follicles. We find that a human SYCP2L intronic single nucleotide polymorphism (SNP) rs2153157, which is associated with ANM, changes the splicing efficiency of U12-type minor introns and may therefore regulate the steady-state amount of SYCP2L transcript. Furthermore, the more efficiently spliced allele of this intronic SNP in SYCP2L is associated with increased ANM. Our results suggest that SYCP2L promotes the survival of primordial oocytes and thus provide functional evidence for its association with ANM in humans.

Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

Respiratory failure, cleft palate and epilepsy in the mouse model of human Xq22.1 deletion syndrome.

Chromosomal segmental deletion is a frequent cause of human diseases. A familial 1.1 Mb deletion of human chromosome Xq22.1 associates with epilepsy, cleft palate and developmental defects in heterozygous female patients. Here, we describe a mouse mutant with a targeted deletion of the syntenic segment of the mouse X chromosome that phenocopies the human syndrome. Male mice with a deletion of a 1.1 Mb Nxf2-Nxf3 X-chromosomal segment exhibit respiratory failure, neonatal lethality and cleft palate. In female mice, heterozygosity for the deletion manifests cleft palate, early postnatal lethality, postnatal growth delay and spontaneous seizures in surviving animals, apparently due to X-chromosome inactivation. Furthermore, loss of a 0.35 Mb subregion containing Armcx5, Gprasp1, Gprasp2 and Bhlhb9 is sufficient to cause the Xq22.1 syndrome phenotype. Our results support that the 1.1 Mb deletion of human Xq22.1 is the genetic cause of the associated syndrome.

Mutations in the MOV10L1 ATP Hydrolysis Motif Cause piRNA Biogenesis Failure and Male Sterility in Mice.

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs. piRNAs protect the genome integrity of the germline by silencing active transposable elements and are essential for germ cell development. Most piRNA pathway proteins are evolutionarily conserved. MOV10L1, a testis-specific RNA helicase, binds to piRNA precursors and is a master regulator of piRNA biogenesis in mouse. Here we report that mutation of the MOV10L1 ATP hydrolysis site leads to depletion of piRNAs on Piwi proteins, de-repression of transposable elements, and conglomeration of piRNA pathway proteins into polar granules. The Mov10l1 mutant mice exhibit meiotic arrest and male sterility. Our results show that mutation of the MOV10L1 ATP hydrolysis site perturbs piRNA biogenesis.