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Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle.sup.1-3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci.sup.4-10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA-RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA-RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term 'DR-loops', which contain both DNA-DNA and DNA-RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops. RNA transcripts stimulate homologous recombination through the formation of DR-loops, intermediate structures that contain both DNA-DNA and DNA-RNA hybrids., Author(s): Jian Ouyang [sup.1] , Tribhuwan Yadav [sup.1] , Jia-Min Zhang [sup.1] , Haibo Yang [sup.1] [sup.2] , Esther Rheinbay [sup.1] , Hongshan Guo [sup.1] [sup.3] , Daniel A. Haber [...]

DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development (1-5). However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements., DNA methylation is an important form of epigenetic modification and has a crucial role in many biological processes, including repression of gene transcription, maintenance of gene imprinting and X-chromosome inactivation, [...]

Study Question: Is it possible to evaluate the methylome of individual oocytes to investigate the DNA methylome alterations in metaphase II (MII) oocytes with reduced embryo developmental potential?Summary Answer: The DNA methylome of each human first polar body (PB1) closely mirrored that of its sibling MII oocyte; hypermethylated long interspersed nuclear element (LINE) and long terminal repeats (LTRs) and methylation aberrations in PB1 promoter regions may indicate poor embryo development.What Is Known Already: The developmental potential of an embryo is determined by the oocyte's developmental competence, and the PB1 is a good substitute to examine the chromosomal status of the corresponding oocyte. However, DNA methylation, a key epigenetic modification, also regulates gene expression and embryo development.Study Design, Size, Duration: Twelve pairs of PB1s and sibling MII oocytes were biopsied and sequenced to compare their methylomes. To further investigate the methylome of PB1s and the potential epigenetic factors that may affect oocyte quality, MII oocytes (n = 74) were fertilized through ICSI, while PB1s were biopsied and profiled to measure DNA methylation. The corresponding embryos were further cultured to track their development potential. The oocytes and sperm samples used in this study were donated by healthy volunteers with signed informed consent.Participants/materials, Setting, Methods: Single-cell methylome sequencing was applied to obtain the DNA methylation profiles of PB1s and oocytes. The DNA methylome of PB1s was compared between the respective group of oocytes that progressed to blastocysts and the group of oocytes that failed to develop. DNA methylation levels of corresponding regions and differentially methylated regions were calculated using customized Perl and R scripts. RNA-seq data were downloaded from a previously published paper and reanalysed.Main Results and the Role Of Chance: The results from PB1-MII oocyte pair validated that PB1 contains nearly the same methylome (average Pearson correlation is 0.92) with sibling MII oocyte. LINE and LTR expression increased markedly after fertilization. Moreover, the DNA methylation levels in LINE (including LINE1 and LINE2) and LTR were significantly higher in the PB1s of embryos that could not reach the blastocyst stage (Wilcoxon-Matt-Whitney test, P < 0.05). DNA methylation in PB1 promoters correlated negatively with gene expression of MII oocyte. Regarding the methylation status of the promoter regions, 66 genes were hypermethylated in the developmental arrested group, with their related functions (significantly enriched in several Gene Ontology terms) including transcription, positive regulation of adenylate cyclase activity, mitogen-activated protein kinase (MAPK) cascade and intracellular oestrogen receptor signalling pathway.Large Scale Data: N/A.Limitations, Reasons For Caution: Data analysis performed in this study focused on the competence of human oocytes and compared them with maternal genetic and epigenetic profiles. Therefore, data regarding the potential regulatory roles of paternal genomes in embryo development are lacking.Wider Implications Of the Findings: The results from PB1-oocyte pairs demonstrated that PB1s shared similar methylomes with their sibling oocytes. The selection of the good embryos for transfer should not only rely on morphology but also consider the DNA methylation of the corresponding PB1 and therefore MII oocyte. The application of early-stage analysis of PB1 offers an option for high-quality oocyte and embryo selection, which provides an additional tool for elective single embryo transfer in assisted reproduction.Study Funding/competing Interest(s): This study was supported by the National Key Research and Development Program of China (2018YFC1004003, 2017YFA0103801), the National Natural Science Foundation of China (81730038, 3187144, 81521002) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020703). The authors have no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc−/−) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the use of 10 000 mouse primordial germ cells for the modification of the chromatin immunoprecipitation-sequencing (ChIP-Seq) of H3K4me3 markers.

In a pilot-scale internal loop airlift reactor with a height of 5.5 m and a main column diameter of 0.484 m, the influence of three gas sparger structures (ladder distributor, tri-nozzle sparger and perforated plate) on the volumetric mass transfer coefficient kLa was investigated. It was found that the perforated plate produces the highest gas holdup difference and circulating liquid velocity between the riser and the downcomer. The perforated plate provides the most efficient mass transfer due to the more uniform gas distribution and higher circulating liquid velocity, followed by the ladder distributor and tri-nozzle spargers. Compared with the tri-nozzle sparger, the perforated plate increases the value of kLa by up to 16% at a superficial velocity of 0.15 m/s. Interestingly, the analysis of the liquid-phase mass transfer coefficient kL and specific area a with respect to gas velocity shows that the mass transfer rate is primarily controlled by a. By comparing the predictions of different mass transfer models, the slip velocity model based on penetration theory yields a satisfactory agreement with the experimental results within ±15% error. Meanwhile, empirical correlations regarding gas holdup and kLa were developed and were found to have good consistency with experimental values. [ABSTRACT FROM AUTHOR]

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3′ UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation. [Display omitted] • NR4A1 specifically localizes to IEG gene bodies suppressing transcriptional elongation • Acute replication stress triggers NR4A1 release, triggering burst of IEG expression • NR4A1 deletion deregulates expression of the IEG FOS , triggering mitotic catastrophe • The NR4A1-IEG pathway denotes differentiated cancers with a favorable prognosis Studying tumorigenesis by patient-derived breast cancer cells, Guo et al. identify NR4A1 as a master regulator of replication stress-induced immediate early gene expression through a transcriptional processing checkpoint. Suppression of NR4A1 in cancer cells dependent on this pathway triggers mitotic catastrophe through unregulated expression of the IEG family member FOS. [ABSTRACT FROM AUTHOR]

SETD1A, a Set1/COMPASS family member maintaining histone-H3-lysine-4 (H3K4) methylation on transcriptionally active promoters, is overexpressed in breast cancer. Here, we show that SETD1A supports mitotic processes and consequentially, its knockdown induces senescence. SETD1A, through promoter H3K4 methylation, regulates several genes orchestrating mitosis and DNA-damage responses, and its depletion causes chromosome misalignment and segregation defects. Cell cycle arrest in SETD1A knockdown senescent cells is independent of mutations in p53, RB and p16, known senescence mediators; instead, it is sustained through transcriptional suppression of SKP2, which degrades p27 and p21. Rare cells escaping senescence by restoring SKP2 expression display genomic instability. In > 200 cancer cell lines and in primary circulating tumor cells, SETD1A expression correlates with genes promoting mitosis and cell cycle suggesting a broad role in suppressing senescence induced by aberrant mitosis. Thus, SETD1A is essential to maintain mitosis and proliferation and its suppression unleashes the tumor suppressive effects of senescence. SETD1A, a histone H3K4 methyltransferase that promotes gene expression, is required for embryonic development. Here, the authors show that SETD1A regulates the expression of mitotic genes and that SETD1A suppression induces senescence. [ABSTRACT FROM AUTHOR]

Summary Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental-stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10 to 11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes. [ABSTRACT FROM AUTHOR]

Background: Patients with relapsed or refractory hematologic cancers have a poor prognosis. Chimeric antigen receptor (CAR) T-cell therapy as a bridge to allogeneic hematopoietic stem-cell transplantation (HSCT) has the potential for long-term tumor elimination. However, pre-HSCT myeloablation and graft-versus-host disease (GVHD) prophylaxis agents have toxic effects and could eradicate residual CAR T cells and compromise antitumor effects. Whether the integration of CAR T-cell therapy and allogeneic HSCT can preserve CAR T-cell function and improve tumor control is unclear., Methods: We tested a novel "all-in-one" strategy consisting of sequential CD7 CAR T-cell therapy and haploidentical HSCT in 10 patients with relapsed or refractory CD7-positive leukemia or lymphoma. After CAR T-cell therapy led to complete remission with incomplete hematologic recovery, patients received haploidentical HSCT without pharmacologic myeloablation or GVHD prophylaxis drugs. Toxic effects and efficacy were closely monitored., Results: After CAR T-cell therapy, all 10 patients had complete remission with incomplete hematologic recovery and grade 4 pancytopenia. After haploidentical HSCT, 1 patient died on day 13 of septic shock and encephalitis, 8 patients had full donor chimerism, and 1 patient had autologous hematopoiesis. Three patients had grade 2 HSCT-associated acute GVHD. The median follow-up was 15.1 months (range, 3.1 to 24.0) after CAR T-cell therapy. Six patients remained in minimal residual disease-negative complete remission, 2 had a relapse of CD7-negative leukemia, and 1 died of septic shock at 3.7 months. The estimated 1-year overall survival was 68% (95% confidence interval [CI], 43 to 100), and the estimated 1-year disease-free survival was 54% (95% CI, 29 to 100)., Conclusions: Our findings suggest that sequential CD7 CAR T-cell therapy and haploidentical HSCT is safe and effective, with remission and serious but reversible adverse events. This strategy offers a feasible approach for patients with CD7-positive tumors who are ineligible for conventional allogeneic HSCT. (Funded by the National Natural Science Foundation of China and the Key Project of Science and Technology Department of Zhejiang Province; ClinicalTrials.gov numbers, NCT04599556 and NCT04538599.)., (Copyright © 2024 Massachusetts Medical Society.)

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF -mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin ( TF ), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities. This article is highlighted in the In This Issue feature, p. 521 ., (©2020 American Association for Cancer Research.)

Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here we performed single-cell RNA-seq analysis of over 2,000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis, and cell-cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of the bone morphogenic protein (BMP) and Notch signaling pathways. Our work provides key insights into the crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present "CLEVER-seq" (chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)