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Cell Genomics Nov 2023Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a...
Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic chromosome , . We designed and built to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics . Overall, we established as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains.
PubMed: 38020970
DOI: 10.1016/j.xgen.2023.100435 -
Fertility and Sterility Jan 2024The oocyte, a long-lived, postmitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout... (Review)
Review
The oocyte, a long-lived, postmitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout reproductive life. Mechanisms of oocyte aging include the accumulation of oxidative damage, mitochondrial dysfunction, and disruption of proteins, including cohesion. Nobel Laureate Bob Edwards also discovered a "production line" during oogonial replication in the mouse, wherein the last oocytes to ovulate in the adult-derived from the last oogonia to exit mitotic replication in the fetus. On the basis of this, we proposed a two-hit "telomere theory of reproductive aging" to integrate the myriad features of oocyte aging. The first hit was that oocytes remaining in older women traversed more cell cycles during fetal oogenesis. The second hit was that oocytes accumulated more environmental and endogenous oxidative damage throughout the life of the woman. Telomeres (Ts) could mediate both of these aspects of oocyte aging. Telomeres provide a "mitotic clock," with T attrition an inevitable consequence of cell division because of the end replication problem. Telomere's guanine-rich sequence renders them especially sensitive to oxidative damage, even in postmitotic cells. Telomerase, the reverse transcriptase that restores Ts, is better at maintaining than elongating T. Moreover, telomerase remains inactive during much of oogenesis and early development. Oocytes are left with short Ts, on the brink of viability. In support of this theory, mice with induced T attrition and women with naturally occurring telomeropathy suffer diminished ovarian reserve, abnormal embryo development, and infertility. In contrast, sperm are produced throughout the life of the male by a telomerase-active progenitor, spermatogonia, resulting in the longest Ts in the body. In mice, cleavage-stage embryos elongate Ts via "alternative lengthening of telomeres," a recombination-based mechanism rarely encountered outside of telomerase-deficient cancers. Many questions about Ts and reproduction are raised by these findings: does the "normal" T attrition observed in human oocytes contribute to their extraordinarily high rate of meiotic nondisjunction? Does recombination-based T elongation render embryos susceptible to mitotic nondisjunction (and mosaicism)? Can some features of Ts serve as markers of oocyte quality?
Topics: Male; Female; Humans; Mice; Animals; Aged; Telomerase; Semen; Reproduction; Aging; Oocytes; Telomere
PubMed: 37993053
DOI: 10.1016/j.fertnstert.2023.11.012 -
Genetics Feb 2024The fourth chromosome is the final frontier for genetic analysis in Drosophila. Small, heterochromatic, and devoid of recombination the fourth has long been ignored....
The fourth chromosome is the final frontier for genetic analysis in Drosophila. Small, heterochromatic, and devoid of recombination the fourth has long been ignored. Nevertheless, its long arm contains 79 protein-coding genes. The Fourth Chromosome Resource Project (FCRP) has a goal of facilitating the investigation of genes on this neglected chromosome. The project has 446 stocks publicly available at the Bloomington and Kyoto stock centers with phenotypic data curated by the FlyBase and FlyPush resources. Four of the five stock sets are nearly complete: (1) UAS.fly cDNAs, (2) UAS.human homolog cDNAs, (3) gene trap mutants and protein traps, and (4) stocks promoting meiotic and mitotic recombination on the fourth. Ongoing is mutagenesis of each fourth gene on a new FRT-bearing chromosome for marked single-cell clones. Beyond flies, FCRP facilitates the creation and analysis of humanized fly stocks. These provide opportunities to apply Drosophila genetics to the analysis of human gene interaction and function. In addition, the FCRP provides investigators with confidence through stock validation and an incentive via phenotyping to tackle genes on the fourth that have never been studied. Taken together, FCRP stocks will facilitate all manner of genetic and molecular studies. The resource is readily available to researchers to enhance our understanding of metazoan biology, including conserved molecular mechanisms underlying health and disease.
Topics: Animals; Humans; Drosophila; Chromosomes; Mutagenesis; Drosophila melanogaster
PubMed: 37981656
DOI: 10.1093/genetics/iyad201 -
Genes To Cells : Devoted To Molecular &... Jan 2024The mitotic cohesin complex necessary for sister chromatid cohesion and chromatin loop formation shows local and global association to chromosomes in response to DNA...
The mitotic cohesin complex necessary for sister chromatid cohesion and chromatin loop formation shows local and global association to chromosomes in response to DNA double-strand breaks (DSBs). Here, by genome-wide binding analysis of the meiotic cohesin with Rec8, we found that the Rec8-localization profile along chromosomes is altered from middle to late meiotic prophase I with cleavage-independent dissociation. Each Rec8-binding site on the chromosome axis follows a unique alternation pattern with dissociation and probably association. Centromeres showed altered Rec8 binding in late prophase I relative to mid-prophase I, implying chromosome remodeling of the regions. Rec8 dissociation ratio per chromosome is correlated well with meiotic DSB density. Indeed, the spo11 mutant deficient in meiotic DSB formation did not change the distribution of Rec8 along chromosomes in late meiotic prophase I. These suggest the presence of a meiosis-specific regulatory pathway for the global binding of Rec8-cohesin in response to DSBs.
Topics: Cell Cycle Proteins; Cohesins; DNA; DNA Breaks, Double-Stranded; Meiosis; Saccharomyces cerevisiae
PubMed: 37968127
DOI: 10.1111/gtc.13081 -
International Journal of Molecular... Oct 2023Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7...
Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7, a mutant that cannot dimerize. The expression of wild-type Rev7, but not the mutant, rescued the sensitivity of Rev7 cells to X-rays and several alkylating agents and reversed the olaparib resistance phenotype of Rev7 cells. Using a novel fluorescent host-cell reactivation assay, we found that Rev7 is unable to promote gap-filling TLS opposite an abasic site analog. The Rev7 dimerization interface is also required for shieldin function, as both Rev7 cells and Rev7 cells expressing Rev7 exhibit decreased proficiency in rejoining some types of double strand breaks, as well as increased homologous recombination. Interestingly, Rev7 retains some function in cell cycle regulation, as it maintains an interaction with Ras-related nuclear protein (Ran) and partially rescues the formation of micronuclei. The mutant Rev7 also rescues the G2/M accumulation observed in Rev7 cells but does not affect progression through mitosis following nocodazole release. We conclude that while Rev7 dimerization is required for its roles in TLS, DSB repair, and regulation of the anaphase promoting complex, dimerization is at least partially dispensable for promoting mitotic spindle assembly through its interaction with Ran.
Topics: Animals; Mice; Anaphase-Promoting Complex-Cyclosome; DNA Repair; DNA Replication; Mad2 Proteins; Mitosis
PubMed: 37958783
DOI: 10.3390/ijms242115799 -
Nucleic Acids Research Dec 2023Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was...
Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was limited by lack of a robust method to construct large monoclonal high-quality combinatorial libraries. This study developed cell cycle arrested recombinase-mediated cassette exchange (aRMCE), able to provide monoclonality, precise genomic integration and uniform transgene expression. Via optimized nocodazole-mediated mitotic arrest, 20% target gene replacement efficiency was achieved without antibiotic selection, and the improved aRMCE efficiency was applicable to a variety of tested cell clones, transgene targets and transfection methods. As a demonstration of this versatile method, we performed directed evolution of fragment crystallizable (Fc), for which error-prone libraries of over 107 variants were constructed and displayed as IgG on surface of CHO cells. Diversities of constructed libraries were validated by deep sequencing, and panels of novel Fc mutants were identified showing improved binding towards specific Fc gamma receptors and enhanced effector functions. Due to its large cargo capacity and compatibility with different mutagenesis approaches, we expect this mammalian cell platform technology has broad applications for directed evolution, multiplex genetic assays, cell line development and stem cell engineering.
Topics: Cricetinae; Animals; Recombinases; Cricetulus; CHO Cells; Transfection; Cell Cycle
PubMed: 37941133
DOI: 10.1093/nar/gkad1001 -
Pathologie (Heidelberg, Germany) Dec 2023In a recently published study a new genetic hypothesis was established that explained the existence of CTNNB1 mutations in Lynch syndrome-associated colorectal... (Review)
Review
In a recently published study a new genetic hypothesis was established that explained the existence of CTNNB1 mutations in Lynch syndrome-associated colorectal carcinomas (MLH1-LS-CRC). This hypothesis states that a mitotic recombination on chromosome 3p simultaneously leads to inactivation of the mismatch repair gene MLH1 and to the activation of CTNNB1. This explains the increased frequency of CTNNB1 mutations in MLH1-LS-CRC compared with other colon carcinomas. To test this hypothesis, various experiments were carried out that show that the first phase of recombination occurs in non-cancerous tissues, which favours the development of CTNNB1 mutations. This mechanism could explain the rapid tumour progression in MLH1-LS-CRC. The results highlight the importance of mitotic recombination in carcinogenesis and provide an insight into the genetic basis of colorectal carcinoma in the context of Lynch syndrome.
Topics: Humans; Colorectal Neoplasms, Hereditary Nonpolyposis; MutL Protein Homolog 1; Colorectal Neoplasms; Promoter Regions, Genetic; Carcinogenesis
PubMed: 37932477
DOI: 10.1007/s00292-023-01245-x -
Seminars in Cell & Developmental Biology Mar 2024Maintenance of genome stability relies on functional centromeres for correct chromosome segregation and faithful inheritance of the genetic information. The human... (Review)
Review
Maintenance of genome stability relies on functional centromeres for correct chromosome segregation and faithful inheritance of the genetic information. The human centromere is the primary constriction within mitotic chromosomes made up of repetitive alpha-satellite DNA hierarchically organized in megabase-long arrays of near-identical higher order repeats (HORs). Centromeres are epigenetically specified by the presence of the centromere-specific histone H3 variant, CENP-A, which enables the assembly of the kinetochore for microtubule attachment. Notably, centromeric DNA is faithfully inherited as intact haplotypes from the parents to the offspring without intervening recombination, yet, outside of meiosis, centromeres are akin to common fragile sites (CFSs), manifesting crossing-overs and ongoing sequence instability. Consequences of DNA changes within the centromere are just starting to emerge, with unclear effects on intra- and inter-generational inheritance driven by centromere's essential role in kinetochore assembly. Here, we review evidence of meiotic selection operating to mitigate centromere drive, as well as recent reports on centromere damage, recombination and repair during the mitotic cell division. We propose an antagonistic pleiotropy interpretation to reconcile centromere DNA instability as both driver of aneuploidy that underlies degenerative diseases, while also potentially necessary for the maintenance of homogenized HORs for centromere function. We attempt to provide a framework for this conceptual leap taking into consideration the structural interface of centromere-kinetochore interaction and present case scenarios for its malfunctioning. Finally, we offer an integrated working model to connect DNA instability, chromatin, and structural changes with functional consequences on chromosome integrity.
Topics: Humans; DNA, Satellite; Centromere; Chromatin; DNA; Meiosis
PubMed: 37926668
DOI: 10.1016/j.semcdb.2023.10.002 -
Genes, Chromosomes & Cancer Jan 2024Smooth muscle tumors are the most common mesenchymal tumors of the female genital tract, including the vulva. Since vulvar smooth muscle tumors are rare, our...
Smooth muscle tumors are the most common mesenchymal tumors of the female genital tract, including the vulva. Since vulvar smooth muscle tumors are rare, our understanding of them compared to their uterine counterparts continues to evolve. Herein, we present two cases of morphologically distinct myxoid epithelioid smooth muscle tumors of the vulva with novel MEF2D::NCOA2 gene fusion. The tumors involved 24 and 37-year-old women. Both tumors presented as palpable vulvar masses that were circumscribed, measuring 2.8 and 5.1 cm in greatest dimension. Histologically, they were composed of epithelioid to spindle-shaped cells with minimal cytologic atypia and prominent myxoid matrix. Rare mitotic figures were present (1-3 mitotic figures per 10 high-power field (HPF)), and no areas of tumor necrosis were identified. By immunohistochemistry, the neoplastic cells strongly expressed smooth muscle actin, calponin, and desmin, confirming smooth muscle origin. Next-generation sequencing identified identical MEF2D::NCOA2 gene fusions. These two cases demonstrate that at least a subset of myxoid epithelioid smooth muscle tumors of the vulva represent a distinct entity characterized by a novel MEF2D::NCOA2 gene fusion. Importantly, recognition of the distinct morphologic and genetic features of these tumors is key to understanding the biological potential of these rare tumors.
Topics: Adult; Female; Humans; Young Adult; Biomarkers, Tumor; Gene Fusion; MEF2 Transcription Factors; Nuclear Receptor Coactivator 2; Smooth Muscle Tumor; Vulva
PubMed: 37870842
DOI: 10.1002/gcc.23209 -
Frontiers in Neuroscience 2023A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the cytoplasmic mislocalization and aggregation of the...
A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the cytoplasmic mislocalization and aggregation of the DNA/RNA-binding protein TDP-43, but how loss of nuclear TDP-43 function contributes to ALS and FTD pathogenesis remains largely unknown. Here, using large-scale RNAi screening, we identify , which encodes TDP-43, as a gene whose loss-of-function results in elevated DNA mutation rate and genomic instability. Consistent with this finding, we observe increased DNA damage in induced pluripotent stem cells (iPSCs) and iPSC-derived post-mitotic neurons generated from ALS patients harboring mutations. We find that the increase in DNA damage in ALS iPSC-derived neurons is due to defects in two major pathways for DNA double-strand break repair: non-homologous end joining and homologous recombination. Cells with defects in DNA repair are sensitive to DNA damaging agents and, accordingly, we find that ALS iPSC-derived neurons show a marked reduction in survival following treatment with a DNA damaging agent. Importantly, we find that increased DNA damage is also observed in neurons with nuclear TDP-43 depletion from ALS/FTD patient brain tissues. Collectively, our results demonstrate that ALS neurons with loss of nuclear TDP-43 function have elevated levels of DNA damage and contribute to the idea that genomic instability is a defining pathological feature of ALS/FTD patients with TDP-43 pathology.
PubMed: 37849894
DOI: 10.3389/fnins.2023.1251228