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Nature Structural & Molecular Biology Oct 2023Long interspersed nuclear element 1 (LINE-1) is the only autonomous retrotransposon in humans and new integrations are a major source of genetic variation between...
Long interspersed nuclear element 1 (LINE-1) is the only autonomous retrotransposon in humans and new integrations are a major source of genetic variation between individuals. These events can also lead to de novo germline mutations, giving rise to heritable genetic diseases. Recently, a role for DNA repair in regulating these events has been identified. Here we find that Fanconi anemia (FA) DNA crosslink repair factors act in a common pathway to prevent retrotransposition. We purify recombinant SLX4-XPF-ERCC1, the crosslink repair incision complex, and find that it cleaves putative nucleic acid intermediates of retrotransposition. Mice deficient in upstream crosslink repair signaling (FANCA), a downstream component (FANCD2) or the nuclease XPF-ERCC1 show increased LINE-1 retrotransposition in vivo. Organisms limit retrotransposition through transcriptional silencing but this protection is attenuated during early development leaving the zygote vulnerable. We find that during this window of vulnerability, DNA crosslink repair acts as a failsafe to prevent retrotransposition. Together, our results indicate that the FA DNA crosslink repair pathway acts together to protect against mutation by restricting LINE-1 retrotransposition.
Topics: Humans; Mice; Animals; Fanconi Anemia; DNA-Binding Proteins; DNA Repair; DNA Damage; Fanconi Anemia Complementation Group D2 Protein; DNA
PubMed: 37580626
DOI: 10.1038/s41594-023-01067-8 -
BMC Biology Jul 2023Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the...
BACKGROUND
Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size.
RESULTS
We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes.
CONCLUSIONS
We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.
Topics: Animals; Humans; Mice; Adenine; HEK293 Cells; Proprotein Convertase 9; RNA, Guide, CRISPR-Cas Systems; Gene Editing
PubMed: 37434184
DOI: 10.1186/s12915-023-01644-9 -
Environmental Health Perspectives Dec 2023Phthalates have been reported to impair fertility in various studies. However, evidence exploring the associations between phthalate metabolites in follicular fluid (FF)...
Associations between Phthalate Metabolite Concentrations in Follicular Fluid and Reproductive Outcomes among Women Undergoing Fertilization/Intracytoplasmic Sperm Injection Treatment.
BACKGROUND
Phthalates have been reported to impair fertility in various studies. However, evidence exploring the associations between phthalate metabolites in follicular fluid (FF) and reproductive outcomes is lacking.
OBJECTIVES
To investigate the associations between phthalate metabolite concentrations in FF and fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes among women recruited from a fertility clinic.
METHODS
We included 641 women undergoing IVF/ICSI treatment from December 2018 to January 2020. The levels of eight phthalate metabolites, including monoethyl phthalate (MEP), mono-isobutyl phthalate (MiBP), mono--butyl phthalate (MBP), monobenzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), were quantified in FF collected on the oocyte retrieval day. Associations between quartiles of individual phthalate metabolite concentrations and nine IVF/ICSI outcomes, including oocyte yield, mature oocyte number, two distinct pronuclei (2PN) zygote number, fertilization rate, blastocyst formation rate, implantation, clinical pregnancy, miscarriage, and live birth, were estimated with generalized linear models. The effects of phthalate mixtures on IVF/ICSI outcomes were assessed using Bayesian kernel machine regression (BKMR) models.
RESULTS
After adjusting for relevant confounders, elevated quartiles of MBzP, MEHHP, and MEHP in FF were inversely associated with the numbers of retrieved oocytes, mature oocytes, and 2PN zygotes (all for trends ). In comparison with the lowest quartile, the highest quartile of molar sum of di(2-ethylhexyl) phthalate metabolites () was associated with a reduction of 9.1% [95% confidence interval (CI): , ] and 10.3% (95% CI: , ) in yielded oocyte and mature oocyte numbers, respectively. Furthermore, the BKMR models revealed inverse associations between phthalate mixtures and the numbers of retrieved oocytes and mature oocytes. We generally found null results for implantation, clinical pregnancy, miscarriage, and live birth.
DISCUSSION
Certain phthalate metabolites in FF are inversely associated with the numbers of retrieved oocytes, mature oocytes, and 2PN zygotes among women undergoing IVF/ICSI treatment. https://doi.org/10.1289/EHP11998.
Topics: Pregnancy; Humans; Male; Female; Sperm Injections, Intracytoplasmic; Abortion, Spontaneous; Follicular Fluid; Bayes Theorem; Environmental Pollutants; Semen; Fertilization in Vitro; Phthalic Acids; Environmental Exposure
PubMed: 38150316
DOI: 10.1289/EHP11998 -
ELife Oct 2023Highly potent animal stem cells either self renew or launch complex differentiation programs, using mechanisms that are only partly understood. female germline stem...
Highly potent animal stem cells either self renew or launch complex differentiation programs, using mechanisms that are only partly understood. female germline stem cells (GSCs) perpetuate without change over evolutionary time and generate cystoblast daughters that develop into nurse cells and oocytes. Cystoblasts initiate differentiation by generating a transient syncytial state, the germline cyst, and by increasing pericentromeric H3K9me3 modification, actions likely to suppress transposable element activity. Relatively open GSC chromatin is further restricted by Polycomb repression of testis or somatic cell-expressed genes briefly active in early female germ cells. Subsequently, Neijre/CBP and Myc help upregulate growth and reprogram GSC metabolism by altering mitochondrial transmembrane transport, gluconeogenesis, and other processes. In all these respects GSC differentiation resembles development of the totipotent zygote. We propose that the totipotent stem cell state was shaped by the need to resist transposon activity over evolutionary timescales.
Topics: Animals; Male; Drosophila; Chromatin; Oogonial Stem Cells; Drosophila Proteins; Stem Cells; Cell Differentiation; Germ Cells; Gene Expression; Biology; Drosophila melanogaster
PubMed: 37831064
DOI: 10.7554/eLife.90509 -
Frontiers in Endocrinology 2023maturation (IVM) of germinal vesicle intact oocytes prior to fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally...
BACKGROUND
maturation (IVM) of germinal vesicle intact oocytes prior to fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation.
OBJECTIVES
To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM.
MATERIALS AND METHODS
The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass.
RESULTS
Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts.
CONCLUSIONS
These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.
Topics: Female; Animals; Cattle; Humans; Melatonin; In Vitro Oocyte Maturation Techniques; Oocytes; Cytogenetic Analysis; Epigenesis, Genetic; Lipids
PubMed: 38027209
DOI: 10.3389/fendo.2023.1280847 -
BioRxiv : the Preprint Server For... Jul 2023The first activation of gene expression during development (zygotic genome activation, ZGA) is accompanied by massive changes in chromosome organization. The connection...
The first activation of gene expression during development (zygotic genome activation, ZGA) is accompanied by massive changes in chromosome organization. The connection between these two processes remains unknown. Using Hi-C for zebrafish embryos, we found that chromosome folding starts by establishing "fountains", novel elements of chromosome organization, emerging selectively at enhancers upon ZGA. Using polymer simulations, we demonstrate that fountains can emerge as sites of targeted cohesin loading and require two-sided, yet desynchronized, loop extrusion. Specific loss of fountains upon loss of pioneer transcription factors that drive ZGA reveals a causal connection between enhancer activity and fountain formation. Finally, we show that fountains emerge in early Medaka and embryos; moreover, we found cohesin-dependent fountain pattern on enhancers of mouse embryonic stem cells. Taken together, fountains are the first enhancer-specific elements of chromosome organization; they constitute starting points of chromosome folding during early development, likely serving as sites of targeted cohesin loading.
PubMed: 37503128
DOI: 10.1101/2023.07.15.549120 -
The Journal of Reproduction and... Jun 2024Totipotency refers to the ability of a single cell to give rise to all the different cell types in the body. Terminally differentiated germ cells (sperm and oocytes)... (Review)
Review
Totipotency refers to the ability of a single cell to give rise to all the different cell types in the body. Terminally differentiated germ cells (sperm and oocytes) undergo reprogramming, which results in the acquisition of totipotency in zygotes. Since the 1990s, numerous studies have focused on the mechanisms of totipotency. With the emergence of the concept of epigenetic reprogramming, which is important for the undifferentiated and differentiated states of cells, the epigenomes of germ cells and fertilized oocytes have been thoroughly analyzed. However, in early immunostaining studies, detailed epigenomic information was difficult to obtain. In recent years, the explosive development of next-generation sequencing has made it possible to acquire genome-wide information and the rise of genome editing has facilitated the analysis of knockout mice, which was previously difficult. In addition, live imaging can effectively analyze zygotes and 2-cell embryos, for which the number of samples is limited, and provides biological insights that cannot be obtained by other methods. In this review, the progress of our research using these advanced techniques is traced back from the present to its earliest years.
Topics: Animals; Mice; Chromatin; Blastocyst; Female; Embryonic Development; Epigenesis, Genetic; Zygote
PubMed: 38462486
DOI: 10.1262/jrd.2023-106 -
Proceedings of the National Academy of... Aug 2023Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic...
Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic RNA-binding sterile-α motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). Here, we resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 1.6 Å resolution, which revealed its composition of a homodimerization D subdomain and a subdomain with similarity to a pseudo-HEAT-repeat analogous topology (PHAT) domain. Furthermore, we show that Smaug directly interacts with the germline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its NTD. We determined the crystal structure of the NTD of Smaug in complex with a Smoothened α-helical peptide to 2.0 Å resolution. The peptide binds within a groove that is formed by both the D and PHAT subdomains. Structural modeling supported by experimental data suggested that an α-helix within the disordered region of Oskar binds to the NTD of Smaug in a mode similar to Smoothened. Together, our data uncover the NTD of Smaug as a peptide-binding domain.
Topics: Animals; Humans; Drosophila; Drosophila Proteins; Germ Cells; Hedgehog Proteins; RNA, Messenger; RNA-Binding Proteins; Repressor Proteins; Receptors, G-Protein-Coupled
PubMed: 37523566
DOI: 10.1073/pnas.2304385120 -
Genome Biology Jul 2023The oocyte-to-embryo transition (OET) converts terminally differentiated gametes into a totipotent embryo and is critically controlled by maternal mRNAs and proteins,...
BACKGROUND
The oocyte-to-embryo transition (OET) converts terminally differentiated gametes into a totipotent embryo and is critically controlled by maternal mRNAs and proteins, while the genome is silent until zygotic genome activation. How the transcriptome, translatome, and proteome are coordinated during this critical developmental window remains poorly understood.
RESULTS
Utilizing a highly sensitive and quantitative mass spectrometry approach, we obtain high-quality proteome data spanning seven mouse stages, from full-grown oocyte (FGO) to blastocyst, using 100 oocytes/embryos at each stage. Integrative analyses reveal distinct proteome reprogramming compared to that of the transcriptome or translatome. FGO to 8-cell proteomes are dominated by FGO-stockpiled proteins, while the transcriptome and translatome are more dynamic. FGO-originated proteins frequently persist to blastocyst while corresponding transcripts are already downregulated or decayed. Improved concordance between protein and translation or transcription is observed for genes starting translation upon meiotic resumption, as well as those transcribed and translated only in embryos. Concordance between protein and transcription/translation is also observed for proteins with short half-lives. We built a kinetic model that predicts protein dynamics by incorporating both initial protein abundance in FGOs and translation kinetics across developmental stages.
CONCLUSIONS
Through integrative analyses of datasets generated by ultrasensitive methods, our study reveals that the proteome shows distinct dynamics compared to the translatome and transcriptome during mouse OET. We propose that the remarkably stable oocyte-originated proteome may help save resources to accommodate the demanding needs of growing embryos. This study will advance our understanding of mammalian OET and the fundamental principles governing gene expression.
Topics: Animals; Mice; Proteome; Transcriptome; Embryo, Mammalian; Blastocyst; Oocytes; Gene Expression Regulation, Developmental; Mammals
PubMed: 37443062
DOI: 10.1186/s13059-023-02997-8 -
BioRxiv : the Preprint Server For... Jan 2024Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its...
Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimer's Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.
PubMed: 38328106
DOI: 10.1101/2024.01.25.577216