Genomic Sites Research Articles

CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma

BackgroundGlioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs.MethodsThe GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice.ResultsWe successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice.ConclusionThe findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.

Evaluation of Long-Term Fermentation Performance with Engineered Saccharomyces cerevisiae Strains

The performance of a microbial fermentation on an industrial scale is subjected to the robustness of the strain. Such strains are genetically engineered to optimize the production of desired compounds in minimal time, but they often fail to maintain high productivity levels for many generations, hindering their effective application in industrial conditions. This study focused on assessing the impact of genomic instability in yeasts that were engineered to produce a fluorescent output by incorporating a reporter gene at one or more genomic locations. The fermentation performance of these strains was evaluated over 100 generations in a sequential batch set-up. In order to bridge the gap between strain engineering and industrial implementation, we proposed the use of novel, host-specific parameters to standardize the strain robustness and evaluate potential improvements. It was observed that yeasts carrying multiple copies of the reporter gene exhibited a more pronounced decrease in output, and the genomic integration site significantly influenced the production. By leveraging these new, host-specific parameters, it becomes possible to anticipate strain behavior prior to incurring substantial costs associated with large-scale production. This approach enhances the economic viability of novel microbial fermentation processes and narrows the divide between laboratory findings and industrial applications.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

Rewiring cancer drivers to activate apoptosis.

Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

QTL mapping and analysis for drought tolerance in rice by genome-wide association study.

Rice drought resistance is a complicated quantitative feature involving a range of biological and agronomic variables, but little is known about the underlying genetics and regulatory mechanisms that regulate drought tolerance. This study used 120 recombinant inbred lines (RILs), derived from a cross between drought tolerant Lvhan 1 and susceptible Aixian 1. The RILs were subjected to drought stress at the first ear stage, and phenotypic data of 16 agronomic and physiological traits under varying conditions were investigated. Genome-wide association study (GWAS) on the drought resistance index of traits was carried out. A total of 9 quantitative trait loci (QTLs) associated with drought-related traits were identified on chromosomes 2, 6, 7, 8, 9, and 10, which includes QTLs for plant height (PH) qPH10.1, effective panicles number (EPN) qEPN6.1, panicle length (PL) qPL9.1, thousand-grain weight (TGW) qTGW2.1, qTGW6.1, qTGW8.1, leaf length (LL) qLL7.1, leaf width (LW) qLW7.1, and leaf area (LA) qLA7.1. The fraction of phenotypic variation explained by individual QTL varied from 10.6% to 13.9%. Except for days to flowering (DTF), the mean values of all traits under normal water management conditions were considerably higher than those under drought conditions. Except for the DTF, the drought resistance index of all rice traits was less than 1, indicating that drought treatment reduced the EPN, FGPP, SSR, PH, and LA, which affected the growth and development of rice. The drought resistance index of DTF was 1.02, indicating that drought prolonged the heading time of rice and diminish the yield parameters. Along with identifying QTLs, the results also predicted ten candidate genes, which are directly or indirectly involved in various metabolic functioning related to drought stress. The identification of these genomic sites or QTLs that effectively respond to water scarcity will aid in the quest of understanding the drought tolerance mechanisms. This study will facilitate the marker-assisted rice breeding and handy in the breeding of drought-tolerant rice varieties.

Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.

Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.

Evaluation of population genetics of Acanthogobius ommaturus using genotyping by sequencing technology

Recent high-throughput genotyping technologies allow for comprehensive genomic analyses on an unprecedented scale. In order to evaluate the pattern of genetic structure, 112 individuals from 4 Acanthogobius ommaturus populations over the coastal waters of China were analyzed by genotyping-by-sequencing method. A total of 48.96 Gb of high-quality sequencing data were obtained, and 19159 SNP loci were identified, 1088 of which were outlier loci. The largest genetic differentiation (Fst) occurred between population Zhoushan and other populations based on all SNP loci and outlier loci. The admixture analysis based on outlier loci showed population Zhoushan formed a separate cluster, while the results based on total SNPs did not show consistent clustering evidence. Additionally, the results of principal component analysis (PCA) showed more obvious grouping information, supporting the above admixture results. Across all populations, the genome-wide distribution patterns for π, He and Tajima’s D were greatly similar. Notably, the range of Tajima’s D values fluctuated sharply, revealing that A. ommaturus genomic sites might be subjected to natural selection. Consequently, together with previous works, the present results further suggested that the genetic differentiation pattern might be caused by biogeographical isolation and the divergent evolution of specific gene sites. The local adaptive evolution may play an important role in the genetic differentiation of population Zhoushan.

A dysfunctional miR-1-TRPS1-MYOG axis drives ERMS by suppressing terminal myogenic differentiation

Rhabdomyosarcoma is the most common pediatric soft tissue tumor, comprising two major subtypes: the PAX3/7-FOXO1 fusion-negative embryonal and the PAX3/7-FOXO1 fusion-positive alveolar subtype. Here, we demonstrate that the expression levels of the transcriptional repressor TRPS1 are specifically enhanced in the embryonal subtype, resulting in impaired terminal myogenic differentiation and tumor growth. During normal myogenesis, expression levels of TRPS1 have to decrease to allow myogenic progression, as demonstrated by overexpression of TRPS1 in myoblasts impairing myotube formation. Consequentially, myogenic differentiation in embryonal rhabdomyosarcoma invitro as well as invivo can be achieved by reducing TRPS1 levels. Furthermore, we show that TRPS1 levels in RD cells, the bona fide model cell line for embryonal rhabdomyosarcoma, are regulated by miR-1 and that TRPS1 and MYOD1 share common genomic binding sites. The myogenin (MYOG) promoter is one of the critical targets of TRPS1 and MYOD1; we demonstrate that TRPS1 restricts MYOG expression and thereby inhibits terminal myogenic differentiation. Therefore, reduction of TRPS1 levels in embryonal rhabdomyosarcoma might be a therapeutic approach to drive embryonal rhabdomyosarcoma cells into myogenic differentiation, thereby generating postmitotic myotubes.

Hidden modes of DNA binding by human nuclear receptors

Human nuclear receptors (NRs) are a superfamily of ligand-responsive transcription factors that have central roles in cellular function. Their malfunction is linked to numerous diseases, and the ability to modulate their activity with synthetic ligands has yielded 16% of all FDA-approved drugs. NRs regulate distinct gene networks, however they often function from genomic sites that lack known binding motifs. Here, to annotate genomic binding sites of known and unexamined NRs more accurately, we use high-throughput SELEX to comprehensively map DNA binding site preferences of all full-length human NRs, in complex with their ligands. Furthermore, to identify non-obvious binding sites buried in DNA–protein interactomes, we develop MinSeq Find, a search algorithm based on the MinTerm concept from electrical engineering and digital systems design. The resulting MinTerm sequence set (MinSeqs) reveal a constellation of binding sites that more effectively annotate NR-binding profiles in cells. MinSeqs also unmask binding sites created or disrupted by 52,106 single-nucleotide polymorphisms associated with human diseases. By implicating druggable NRs as hidden drivers of multiple human diseases, our results not only reveal new biological roles of NRs, but they also provide a resource for drug-repurposing and precision medicine.

High-throughput library transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS)

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein–protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here, we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from predefined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

High-throughput library transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS).

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here, we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from predefined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

Random Integration Analysis of Recombinant Adeno-Associated Virus 6 Packaged in Sf9 Insect Cells

Recently, there have been growing concerns over the integration of recombinant adeno-associated virus (rAAV) used in gene therapy. Wild-type adeno-associated virus (AAV) site specifically integrates into AAVS1 site of human genome, while rAAV randomly integrates into host chromosomes at low frequencies. This research aims to study the random integration events of rAAV6-EGFP packaged in Sf9 insect cells. Baculo-Sf9 manufacturing platform has the advantages of high-density suspension culture of Sf9 insect cells and large-scale production of rAAV vectors. In this study, we used different doses of Baculo-Sf9 produced rAAV6-EGFP to transduce HEK293T cells and A549-implanted tumors in vitro and in vivo. Using flow cytometry and fluorescence microscopy, we studied their EGFP gene expression efficiencies and EGFP fluorescence intensities. Using inverse nested PCR and DNA sequencing, random integration sites of rAAV6-EGFP genome into human chromosomes were identified. In vitro results showed that gene expression efficiencies became stable after 20 days and random integration frequencies were 0.2‒4.2%. Both in vitro and in vivo results indicated that random integration of Baculo-Sf9 rAAV6 was dose-dependent. Sequencing results showed two random integration sites, which were on human chromosomes 8 and 12. The findings suggest that we should use as low dose of rAAV vector as possible for safe gene therapy.

Readout of histone methylation by Trim24 locally restricts chromatin opening by p53

The genomic binding sites of the transcription factor (TF) and tumor suppressor p53 are unusually diverse with regard to their chromatin features, including histone modifications, raising the possibility that the local chromatin environment can contextualize p53 regulation. Here, we show that epigenetic characteristics of closed chromatin, such as DNA methylation, do not influence the binding of p53 across the genome. Instead, the ability of p53 to open chromatin and activate its target genes is locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated histone 3 lysine 4 (H3K4), thereby preferentially localizing to those p53 sites that reside in closed chromatin, whereas it is deterred from accessible chromatin by H3K4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to affect gene expression as a function of the local chromatin state. These findings link H3K4 methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors that locally modulate TF function.

O-075 Deficiency of DNA double-strand break repair in human preimplantation embryos revealed by CRISPR-Cas9

Abstract Study question Is double-strand DNA break repair functional in human preimplantation embryos and what implications might a deficiency have for genome editing performed at early developmental stages? Summary answer CRISPR-Cas9 succeeds in generating DNA breaks with high efficiency, but these often remain unresolved, indicating that early human embryos are deficient in DNA repair. What is known already Genome editing (GE) technologies, such as CRISPR-Cas9, raise the possibility of avoiding transmission of inherited disorders by converting mutant genes back to wild-type (normal). The therapeutic application of CRISPR-Cas9 at the preimplantation stage is controversial due to uncertainty over safety and concerns about the alteration of the human germline. However, from a technical perspective it remains highly attractive, since it is the only developmental stage where delivery of CRISPR-Cas9 to every cell of the individual can be guaranteed. Currently, information on safety aspects and efficacy are lacking. Therefore, evaluation of the cellular response to CRISPR-Cas9 in human embryos is needed. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks (DSBs) were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods CRISPR-Cas9 components targeted several non-coding sites in the genome. An artificial DNA fragment was provided, which cells could potentially use for homology directed repair (HDR). Embryos were grown for 60 hours, then disaggregated. Blastomeres underwent whole genome amplification, followed by next generation sequencing to detect segmental aneuploidy related to failure of DNA repair. Additionally, sites targeted by CRISPR-Cas9 were amplified and sequenced to confirm whether repair had occurred and to distinguish the specific mechanism employed. Main results and the role of chance The study successfully validated and utilised three CRISPR ‘guides’ that targeted sites on chromosomes 2, 3 and 12. Alterations at the targeted sites were detected in 24/25 embryos (96% targeting efficiency). Repair of DSBs created by CRISPR-Cas9 was considered successful when an embryo contained at least one blastomere with changes to the DNA sequence at the targeted site consistent with nonhomologous end joining (NHEJ) or HDR. In total, 53 double-stranded breaks were generated by CRISPR-Cas9 during this study. Of these, 51% (27) were repaired by NHEJ, 9% (5) repaired by HDR and 40% (21) remained unresolved, the persistent breakage eventually causing segmental aneuploidy with a breakpoint at the targeted side. Segmental abnormalities are known to be detrimental to embryonic viability and risk congenital abnormalities in offspring. This study shows that human embryos, prior to activation of the genome, have a DNA damage repair deficiency. Timings of morphokinetic milestones were altered in embryos with unresolved DNA damage, confirming that checkpoint control is active. However, continued mitotic division, despite the presence of DSBs, shows cellular mechanisms that usually preserve genetic integrity lack stringency, ultimately failing to ensure repair. The results provide a warning against the therapeutic use of CRISPR-Cas9 in human embryos. Limitations, reasons for caution While targeting the embryonic genome using CRISPR-Cas9 was remarkably efficient (DSBs introduced in 96% of embryos), the majority of cells utilised NHEJ for repair, a process allowing introduction of additional mutations rather than correcting existing ones. Only <10% of cells used the HDR repair mechanism necessary for removal of mutations. Wider implications of the findings These findings reveal that cells of early human embryos are less able to process DSBs than other cell types. This suggests that embryo culture methods should give consideration to protecting the genome from damage. The results caution against clinical application of genome editing technologies that create DSBs, such as CRISPR-Cas9. Trial registration number NA

P-287 Morphokinetic analysis of early embryos subjected to double stranded DNA damage provides evidence for cell cycle checkpoint activity

Abstract Study question Is there evidence of cell cycle checkpoint activation in preimplantation embryos with double stranded breaks (DSBs) in their DNA, induced using CRISPR? Summary answer Cells with persistent double strand DNA damage exhibit longer timeframes to reach key morphological milestones than those with successful repair, consistent with checkpoint activation. What is known already In most cell types, DNA damage leads to activation of cell cycle checkpoints that halt the cycle while DNA repair is attempted. It has been hypothesised that early embryos lack (or have relaxed) checkpoints, and that this may be necessary to allow the embryo to divide rapidly and synchronously during the first few mitoses. It is thought that maternal (oocyte derived) inhibitors may be responsible for suppressing checkpoints until activation of the embryonic genome. Inadequate checkpoint control may be responsible for the genetic instability and sensitivity to DNA damage seen during early preimplantation stages, which is of clinical importance. Study design, size, duration 84 embryos were generated for research in an IRB approved study. For this purpose, donor oocytes were fertilised with donor sperm using ICSI. 51 of the resulting embryos served as controls, while in the other 33 double strand DNA breaks were created in a highly controlled fashion, directed at specific genomic sites using CRISPR-Cas9 technology. Successfully fertilised oocytes underwent culture in a time-lapse incubator and the duration of key developmental events were carefully timed. Participants/materials, setting, methods Precise induction of DNA damage involved injection of CRISPR-Cas9 ribonuclear complex (RNP), along with the sperm at the time of fertilization (ICSI). All embryos were disaggregated on day-3 and their cells subjected to whole genome amplification. Amplified products underwent low-pass next generation sequencing to detect segmental aneuploidy related to failure of DNA repair, while the site targeted using CRISPR-Cas9 was PCR amplified and sequenced to confirm whether it had successfully undergone repair. Main results and the role of chance Embryos that successfully repaired the induced DNA damage showed timings of pronuclei appearance/disappearance and cell divisions that were indistinguishable from control embryos. Rates of embryo arrest prior to completion of the second mitotic division were also similar between the two groups (25% versus 24%). In contrast, embryos with unresolved DNA damage, as evidenced by the detection of chromosomal fragments involving a breakpoint at the site targeted using CRISPR, were delayed in reaching the same developmental milestones (p < 0.0001 for time to first cleavage division) and displayed a much higher incidence of arrest (63% before the second mitotic division; p = 0.0002). The delay in mitotic progression in blastomeres with DNA damage represents strong evidence that a checkpoint is active in the cells of cleavage stage embryos, sensing double strand DNA breaks and slowing the cell cycle. However, the fact that 36% of embryos with unresolved DNA damage continued to progress, albeit at a slower rate, is consistent with the notion that checkpoint control is less stringent in early human embryos. This relaxation of control is likely to contribute to the genetic instability seen at the cleavage stage, including a risk of unresolved DNA damage and a high frequency of chromosomal malsegregation (causing mosaicism). Limitations, reasons for caution While altered cell cycle timings are consistent with checkpoint activation, other factors could conceivably influence the duration of key morphokinetic events. Complimentary experiments to test the functionality of specific checkpoints and/or the activity of their individual components will be required for definitive proof that embryonic checkpoints are active but weakened. Wider implications of the findings Segmental aneuploidy, micronucleation and mosaicism are frequently seen in human preimplantation embryos and are associated with reduced likelihood of ongoing pregnancy. Such abnormalities are symptomatic of genetic instability associated with excessively permissive checkpoints. These results highlight the need for culture systems that protect embryos from cellular stressors, especially DNA damage. Trial registration number N.A.

Distant sequence regions of JBP1 contribute to J-DNA binding.

Base-J (β-D-glucopyranosyloxymethyluracil) is a modified DNA nucleotide that replaces 1% of thymine in kinetoplastid flagellates. The biosynthesis and maintenance of base-J depends on the base-J-binding protein 1 (JBP1) that has a thymidine hydroxylase domain and a J-DNA-binding domain (JDBD). How the thymidine hydroxylase domain synergizes with the JDBD to hydroxylate thymine in specific genomic sites, maintaining base-J during semi-conservative DNA replication, remains unclear. Here, we present a crystal structure of the JDBD including a previously disordered DNA-contacting loop and use it as starting point for molecular dynamics simulations and computational docking studies to propose recognition models for JDBD binding to J-DNA. These models guided mutagenesis experiments, providing additional data for docking, which reveals a binding mode for JDBD onto J-DNA. This model, together with the crystallographic structure of the TET2 JBP1-homologue in complex with DNA and the AlphaFold model of full-length JBP1, allowed us to hypothesize that the flexible JBP1 N-terminus contributes to DNA-binding, which we confirmed experimentally. Α high-resolution JBP1:J-DNA complex, which must involve conformational changes, would however need to be determined experimentally to further understand this unique underlying molecular mechanism that ensures replication of epigenetic information.

SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity

Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ–SMAD signaling axis.

Integrator facilitates RNAPII removal to prevent transcription-replication collisions and genome instability

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.

Endometrial carcinoma: association between mutational status, sites of metastasis, recurrence, and correlation with overall survival.

To investigate the association between sites of endometrial carcinoma (EC) recurrence and metastases, mutational status, race, and overall survival (OS). This single-center retrospective study evaluated patients with biopsy-proven EC that underwent genomic molecular testing between January 2015 and July 2021. Association between genomic profile and sites of metastases or recurrence was performed using Pearson's chi-squared or Fisher exact test. Survival curves for ethnicity and race, mutations, sites of metastases or recurrence were estimated using the Kaplan-Meier method. Univariable and multivariable Cox proportional hazard regression models were used. The study included 133 women [median age 64 years (IQR 57-69)]. The most common mutation was TP53 (65/105 patients, 62%). The most common site of metastasis was the peritoneum (35/43, 81%). The most common recurrence was in lymph nodes (34/75, 45%). Mutations of TP53 and PTEN were significantly associated with Black women (p = 0.048, p = 0.004, respectively). In the univariable Cox regression analyses, TP53 mutation and presence of recurrence or metastases to the peritoneum were associated with lower OS (HR 2.1; 95% CI 1.1, 4.3; p = 0.03/ HR 2.9; 95% CI 1.6, 5.4; p = 0.0004; respectively). On multivariable Cox proportional hazards model ER expression (HR 0.4; 95% CI 0.22, 0.91; p = 0.03), peritoneal recurrence or metastases (HR 3.55; 95% CI 1.67, 7.57; p = 0.001), and Black race (HR 2.2; 95% CI 1.1, 4.6; p = 0.03) were significant independent predictors of OS. The integration of EC mutational status and clinicopathological risk assessment demonstrated potential implications on the patterns of metastasis, recurrence, and OS.

Transgenerational sperm DMRs escape DNA methylation erasure during embryonic development andepigenetic inheritance.

Germline transmission of epigenetic information is a critical component of epigenetic inheritance. Previous studies have suggested that an erasure of DNA methylation is required to develop stem cells in the morula embryo. An exception involves imprinted genes that escape this DNA methylation erasure. Transgenerational differential DNA methylation regions (DMRs) have been speculated to be imprinted-like and escape this erasure. The current study was designed to assess if morula embryos escape the erasure of dichlorodiphenyltrichloroethane-induced transgenerational sperm DMR methylation. Observations demonstrate that the majority (98%) of transgenerational sperm DMR sites retain DNA methylation and are not erased, so appearing similar to imprinted-like sites. Interestingly, observations also demonstrate that the majority of low-density CpG genomic sites had a significant increase in DNA methylation in the morula embryo compared to sperm. This is in contrast to the previously observed DNA methylation erasure of higher-density CpG sites. The general erasure of DNA methylation during embryogenesis appears applicable to high-density DNA methylation sites (e.g. CpG islands) but neither to transgenerational DMR methylation sites nor to low-density CpG deserts, which constitute the vast majority of the genome's DNA methylation sites. The role of epigenetics during embryogenesis appears more dynamic than the simple erasure of DNA methylation.