Short G1 Phase Research Articles

Dimethyl Sulfoxide Attenuates Ionizing Radiation-induced Centrosome Overduplication and Multipolar Cell Division in Human Induced Pluripotent Stem Cells.

Centrosomes are important organelles for cell division and genome stability. Ionizing radiation exposure efficiently induces centrosome overduplication via the disconnection of the cell and centrosome duplication cycles. Over duplicated centrosomes cause mitotic catastrophe or chromosome aberrations, leading to cell death or tumorigenesis. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into all organs. To maintain pluripotency, PSCs show specific cellular dynamics, such as a short G1 phase and silenced cell-cycle checkpoints for high cellular proliferation. However, how exogenous DNA damage affects cell cycle-dependent centrosome number regulation in PSCs remains unknown. This study used human iPSCs (hiPSCs) derived from primary skin fibroblasts as a PSC model to address this question. hiPSCs derived from somatic cells could be a useful tool for addressing the radiation response in cell lineage differentiation. After radiation exposure, the hiPSCs showed a higher frequency of centrosome overduplication and multipolar cell division than the differentiated cells. To suppress the indirect effect of radiation exposure, we used the radical scavenger dimethyl sulfoxide (DMSO). Combined treatment with radiation and DMSO efficiently suppressed DNA damage and centrosome overduplication in hiPSCs. Our results will contribute to the understanding of the dynamics of stem cells and the assessment of the risk of genome instability for regenerative medicine.

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration.

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, we investigate models of compensatory proliferation in Drosophila imaginal discs. We find that compensatory proliferation is associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models.

Atypical cell cycle profile of mouse embryonic stem cell is regulated by classic oncogenic and tumor suppressive genes in vitro

Embryonic stem cells (ESCs) exhibit an unusual cell cycle profile containing a short G1 phase. Whether this feature is required to maintain pluripotency is a matter of debate. Here, we report that the short G1 phase is a consequence of MEK1/2 kinase-mediated promotion of G1/S transition, but not necessarily coupled with pluripotency maintenance. We find that compared to primed ESCs, naïve ESCs exhibit a significantly longer G1 phase due to the inhibition of MEK1/2 kinases. MEK1/2 inhibition increases intracellular level of reactive oxygen species (ROS), leading to the stabilization of p53 protein. The genetic ablation of p53 largely converts the cell cycle profile of naïve ESCs to that of primed ESCs. These results demonstrate that pluripotency and proliferation are separable cellular events, and the short G1 phase of primed ESCs is a manifestation of the intricate interplay between classical oncogenes MEK1/2 and tumor suppressor gene TP53 to promote G1/S transition.

Cell Cycle and Differentiation Arrest in Senescent Population of Human Hematopoietic Stem and Progenitor Cell Compartment

Background: Aging is characterized by accumulation of senescent cells in the hematopoietic stem and progenitor cell (HSPC) compartment. Our previous studies have demonstrated that these senescence HSPCs are associated with elevated central carbon metabolism. In this study, we have defined the functional properties of these senescent HSPC. Methods: HSPCs (CD34+ cells) were isolated from the bone marrow of healthy human subjects (range: 20 - 74 years; n = 18; old defined as >60 years and young defined as between 19 to 35 years) and separated by FACS in three distinct subpopulations with high, medium, and low glucose uptake (GU) capacity (GUhigh, GUmed, GUlow). Single-cell RNA-sequencing (scRNAseq) studies, normalization and clustering of the sequencing data, cell cycle regression (to excludes changes due to cell cycle), gene ontology (GO) of biological processes, gene set enrichment analysis (GSEA), pseudotime ordering using Monocle 3, and developmental trajectory analyses were performed. Results: Our previous studies revealed that the GUhigh subset is coupled with significantly higher expression of genes involved in myeloid development, cell cycle arrest, senescence associated secretory phenotype (SASP), anti-apoptosis and survival pathways, and significantly lower expressions of genes involved in lymphoid development. Single cell transcriptomic studies showed that the GUhigh and GUmed subsets consist mainly of HSPCs involved in myeloid development and GUlow subset in lymphoid development. In this study, we have compared the differential gene expressions between old versus young subjects in each of the three subsets with distinct glucose metabolic levels: GUhigh, GUmed and GUlow. The major findings from GSEA (using the gene sets: Hallmarks, Wikipathways, Reactomes, and KEGG) that are relevant to cellular senescence are summarized in Figure 1. These results confirm that the deregulations inherent in senescent cells are found mainly in the GUhigh subset of elderly human subjects. Comparing the frequencies of old versus young GUhigh cells along the cell cycle progression ranks showed a significant reduction of cells in the late G1 phase among GUhigh cells. The differentiation trajectories of HSPCs between old and young human subjects, especially of the GUhigh subset, develop differently (Fig. 1B). When comparing the trajectories of the GUhigh subsets (i.e. senescent HSPCs) between old versus young subjects, we found a significant increase in upregulation of genes inherent in non-primed HSPCs (ADGRG6, HLF, PTPRC) and significant down regulation of genes characteristic for primed HSPCs (HBB, GYPC, CSF2RB, CD74, CD47, LAIR1) in the elderly. Conclusions: Our series of proteome and single cell transcriptome analyses have demonstrated that the GUhigh subpopulation is highly enriched in senescent HSPCs. Cell cycle analysis has revealed a significantly shortened G1 phase in these senescent cells. Developmental trajectories have demonstrated that these cells are in dedifferentiated state. Above all, we have provided evidence for a dependency of senescent HSPCs on elevated glucose metabolism and on anti-apoptotic factors for survival. These mechanisms may represent targets for elimination of senescent HSPCs. Legend for Figure 1: A. Results of Gene Set enrichment analysis (GSEA) comparing the GUhigh subsets of CD34+ cells derived from old versus young human subjects (in first column).The enrichment scores (x102) (ES) are reflected in the size of the circles and the statistical significance (p-values) in the color scale of the circles. Negative ES, i.e. enriched in the GUhigh of young adults, are found in "Mitotic spindle", "DNA repair", "B cell receptor signaling pathway", and in "Apoptosis". The corresponding comparisons between old versus young subjects for GUmed and GUlow subsets are depicted in the second and third columns. B. Developmental trajectories of the HSPCs derived from old versus young human subjects. 1 = early progenitor cells, E = erythroid cells, Meg = megakaryocytes, G = granulocytes, Ly = lymphoid cells. HSPCs, especially the GUhigh subset, from the elderly have a distinctly different developmental trajectory than the corresponding subset from the young. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunohistochemical Expression of Cyclin D1 in Oral Lichen Planus

Background: Many studies have reported that a condition initially classified as oral lichen planus (OLP) has a variable chance of developing cancer progression over time, although these results are still questionable. Cyclin D1 controls the mitotic cell cycle's transition from G1 to S. Cyclin D1 has a significant function in carcinogenesis due to its critical involvement in cell cycle control. Cyclin D1 deregulation or overexpression may result in shortened G1 phase, enhanced cell replication, and decreased reliance on growth regulators, causing disruption in conventional cell cycle progression and the progression of cancer. The aim of the current study was to evaluate the expression of Cyclin D1 in histopathologically confirmed OLP samples and to predict the rate of malignant transformation in these lesions by comparing the level of expression with some clinical parameters. Materials and Methods: The immunohistochemical procedure was used to examine the expression of cyclin D1 on paraffin-embedded sections of (40) OLP lesions and (10) specimens of normal oral mucosa. We used the SPSS software version (18.0) to do the statistical analysis in this work. Results: There is a very significant difference between the people investigated (P = 0.01), which is statistically significant. CyclinD1 was found in all of the oral lichen planus cases in the research (100%), but it was also found (80%) in the normal oral mucosa. There was also no statistically significant difference between the clinical types of OLP and the Cyclin D1 scoring (P = 0.942), which revealed that the reticular type was the most frequent (7) in mild scoring, whereas the erosive type was the most frequent in severe scoring, which accounted for the majority of the cases. Conclusion: This research found that higher cyclinD1 expression increases the risk of cancer and many of the markers should be tested. Furthermore, larger sample numbers, improved designs, and gene-reduction studies should be performed. OLP patients should be continuously monitored for OSCC progression.

Immuno-senescence and elevated central carbon metabolism in hematopoietic stem cell compartment upon aging.

e24030 Background: Aging is associated with decline of hematopoiesis, compromised innate immunity, and propensity for malignancies. Aging of the hematopoietic stem and progenitor cell (HSPC) compartment is caused by accumulation of senescent cells and elimination of the latter rejuvenates innate immunity in animal models. Methods: HSC (CD34+) were isolated from the bone marrow of healthy human subjects (age range 20 – 74 years; n = 18) and separated by FACS in 3 distinct subsets with high, intermediate, and low glucose uptake (GU) capacity (GUhigh, GUinter, GUlow). Single-cell RNA-sequencing (scRNAseq) studies, followed by gene ontology (GO) analysis of biological processes, analysis of cell cycle, and gene set enrichment analysis (GSEA) were performed. Results: While the percentages of GUinter and GUlow subpopulations remain relatively constant with age, the proportion of GUhigh subset increases continuously with age (from 0.2% in adults < 30 years to 6,7% in > 60 years). GO analysis revealed that the GUhigh subset showed a significantly higher expression of genes involved in myeloid development, cell cycle arrest, inflammation and stress response, apoptosis, and significantly lower expressions of genes involved in lymphoid development. GSEA confirmed that the gene sets for cell cycle arrest, senescence associated secretory phenotype (SASP), anti-apoptosis/survival and P53 pathways are significantly up-regulated in the GUhigh subset. The latter shows therefore all deregulations that are inherent hallmarks of senescence. Most of the molecular deregulations are similar to those found in murine models of aging, but there are significant differences. In addition, cell cycle analysis revealed that the proportion of cells in the G1 phase was significantly reduced in the GUhigh subset. Comparing the frequencies of old to young GUhigh, GUlow cells along the cell cycle progression ranks showed a significant depletion of cells in late G1 phase among GUhigh cells but not in the other subsets. The frequencies of non-cycling cells in the GUlowsubset have remained high (> 70%) irrespective of age, thus providing evidence for the quiescence status of the GUlow subset, as well as for persistence of HSPC with “young phenotype”. Conclusions: Our series of proteome and single cell transcriptome analyses have shown that the GUhigh subpopulation is highly enriched in senescent HSPCs. This property may be exploited to enrich, visualize, and trace senescence in human bone marrow. Cell cycle analysis has revealed a significantly shortened G1 phase that is linked to dedifferentiated state in the senescent HSPC. Our results indicate a dependency of senescent HSPCs on elevated glucose metabolism as well as on anti-apoptotic factors for survival. These may represent ideal targets for elimination of senescent HSPC to restore innate immunity as demonstrated in murine models.

Intrinsic neural stem cell properties define brain hypersensitivity to genotoxic stress

SummaryImpaired replication has been previously linked to growth retardation and microcephaly; however, why the brain is critically affected compared with other organs remains elusive. Here, we report the differential response between early neural progenitors (neuroepithelial cells [NECs]) and fate-committed neural progenitors (NPs) to replication licensing defects. Our results show that, while NPs can tolerate altered expression of licensing factors, NECs undergo excessive replication stress, identified by impaired replication, increased DNA damage, and defective cell-cycle progression, leading eventually to NEC attrition and microcephaly. NECs that possess a short G1 phase license and activate more origins than NPs, by acquiring higher levels of DNA-bound MCMs. In vivo G1 shortening in NPs induces DNA damage upon impaired licensing, suggesting that G1 length correlates with replication stress hypersensitivity. Our findings propose that NECs possess distinct cell-cycle characteristics to ensure fast proliferation, although these inherent features render them susceptible to genotoxic stress.

Studying Stem Cell Biology in Intact and Whole-Body Regenerating Hydra by Flow Cytometry.

The freshwater Hydra polyp is a versatile model to study whole-body regeneration from a developmental as well as a cellular point of view. The outstanding regenerative capacities of Hydra are based on its three populations of adult stem cells located in the central body column of the animal. There, these three populations, gastrodermal epithelial, epidermal epithelial, and interstitial, continuously cycle in homeostatic conditions, and their activity is locally regulated after mid-gastric bisection. Moreover, they present an unusual cycling behavior with a short G1 phase and a pausing in G2. This particular cell cycle has been studied for a long time with classical microscopic methods. We describe here two flow cytometry methods that provide accurate and reproducible quantitative data to monitor cell cycle regulation in homeostatic and regenerative contexts. We also present a cell sorting procedure based on flow cytometry, whereby stem cells expressing a fluorescent reporter protein in transgenic lines can be enriched for use in applications such as transcriptomic, proteomic, or cell cycle analysis.

DNA Damage Response and Cell Cycle Regulation in Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including FOXM1, SIRT1 and PUMA. They function through transcription regulation of downstream targets (P53, CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of FOXM1 and SIRT1 on maintaining genomic integrity.

MiR-302 family, miR-145 and miR-296 temporal expression profile along the cell cycle of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), like embryonic (hESCs) and induced pluripotent stem cells (hiPSCs), exhibit an unusual cell cycle structure characterized by a short G1 phase and cells being most of time in S phase. hPSCs are receptive to differentiation cues during their transition through G1 phase when lineage determination is decided. Although several MicroRNAs (miRNAs) have been shown to target transcripts that directly or indirectly coordinate the cell cycle of pluripotent cells, its temporal expression profile along hPSCs cell cycle remains poorly characterized. miR-145 and miR-296 are induced during differentiation and silence the self-renewal and pluripotency program. miR-302 family is essential for hPSCs stemness and its expression decreases during differentiation. We aimed to study how the aforementioned miRNAs are regulated along the cell cycle of hPSCs. We demonstrated by pharmacological synchronization and block and release experiments that miR-145, miR-296 and miR-302 family are periodically expressed in hPSCs. Importantly, miR-302 family expression is induced at G1/S boundary and remained high at S phase, presumably to impede differentiation onset. Besides, we confirmed by a gene ontology analysis that many validated miR-302 family target genes are involved in cell cycle regulation.

Loss of Mediator complex subunit 13 (MED13) promotes resistance to alkylation through cyclin D1 upregulation.

Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR–Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.

Nuclear control of early embryonic development in domestic pigs

In mammals, growing oocytes have characteristically high levels of RNA synthesis. After the initiation of meiosis, that is germinal vesicle breakdown, this RNA synthesis ceases. Although there is limited evidence for RNA synthesis by the zygote, significant amounts of RNA synthesis do not occur until a species-specific cell stage. In pigs, significant amounts of mRNA synthesis cannot be detected before the four-cell stage. There appear to be three qualitatively different periods of transcription during the four-cell stage. The first occurs during a short (< 2 h) G1 phase. The second, occurs after completion of DNA synthesis (S phase) about 16 h after cleavage to the four-cell stage, and the third occurs about 24 h after cleavage to the four-cell stage. Correlated with these changes in RNA synthesis are changes in nucleolar morphology, amino acid transport characteristics, protein production, mitochondrial morphology, and metabolism of the embryo. The mechanisms that regulate initiation of RNA synthesis in early mammalian embryos appear to repress transcription. A state of transcription permissiveness then follows that sets into motion the differentiation programme.

Corrigendum.

Development, Growth & DifferentiationVolume 62, Issue 6 p. 462-462 CORRIGENDUMFree Access Corrigendum This article corrects the following: Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming Asuka Takehara, Yasuhisa Matsui, Volume 61Issue 6Development, Growth & Differentiation pages: 357-364 First Published online: June 14, 2019 First published: 26 August 2020 https://doi.org/10.1111/dgd.12672AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Takehara and Mtasui (2019), the authors note that, in the abstract, a sentence ‘We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1-S transition and is transcriptionally repressed by p53, was significantly downregulated by AKT activation.’ should be ‘We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1-S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation.’ The authors would like to apologize for any inconvenience caused. REFERENCE Takehara, A, & Mtasui, Y (2019). Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming. Development Growth and Differentiation, 61(6), 357– 364. https://doi.org/10.1111/dgd.12621 Volume62, Issue6August 2020Pages 462-462 ReferencesRelatedInformation

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

CHL1 promotes insulin secretion and negatively regulates the proliferation of pancreatic β cells

Cell adhesion molecule L1-like protein (CHL1) is a member of neural recognition molecules of immunoglobulin superfamily primarily expressing in the nervous system. CHL1 regulates neuronal migration, axonal growth, and dendritic projection. Downregulation of CHL1 has been reported in β cells of patients with type 2 diabetes (T2DM). However, the detailed role of CHL1 in β cells has not been characterized. In this study, Real-Time PCR and Western blot were applied to investigate the tissue/cell distribution and expression of CHL1. Gain- or loss-of function studies were conducted in MIN6 cells to determine the effects of CHL1 on cell proliferation, apoptosis, cell cycle, and insulin secretion. Following silencing of CHL1 in MIN6 cells (si-CHL1), insulin secretion and the number of insulin secretary granules <50 nm from the cell membrane decreased in response to 20 mM glucose. Besides, silencing of CHL1 induced cell proliferation, reduced apoptosis, and prolonged S phase and shortened G1 phase of the cell cycle, contrary to overexpressing of CHL1. The inhibitor of ERK1/2MAPK eliminated the effect of CHL1 deficiency on the proliferation of MIN6 cells. In addition, high-fat diet could result in increased islet volume and β cell proliferation, decreased CHL1 expression and activation of ERK pathway in mice islets. Consequently, CHL1 expression was decreased in islets of high-fat induced mice, which resulted in cell proliferation via ERK pathway and regulation of the cell cycle through p53 pathway. These mechanisms may contribute to pancreatic β cell compensatory hyperplasia in obesity-induced pre-diabetes.

Critical Role for P53 in Regulating the Cell Cycle of Ground State Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1 phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here, we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and that is required for the elongated G1 phase characteristic of ground state ESCs. RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that P53 directly regulates the expression of the retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveal a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends onMUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Three-dimensional microenvironmental priming of human mesenchymal stem cells in hydrogels facilitates efficient and rapid retroviral gene transduction via accelerated cell cycle synchronization

There are numerous approaches to improve the low transduction efficiency of retroviral vectors in two-dimensional (2D) cell culture substrates. However, the effect of a three-dimensional (3D) microenvironment, which better mimics in vivo conditions, is unknown. Cytocompatible hyaluronic acid (HA) hydrogels are a good candidate to study this issue. Here, photocrosslinkable HA hydrogels with an elastic modulus of 1.0–2.7 kPa were successfully prepared by varying the degree of methacrylation in the HA backbone. Culturing human adipose-derived stem cells (hASCs) in a 3D microenvironment significantly reduces the amount of time required for retroviral gene transduction compared with the conventional 2D method and maintains a high transduction efficiency. This acceleration of retroviral gene transduction correlates with the rate of cell-cycle synchronization. hASCs cultured in a 3D microenvironment have a shorter G1 phase and total cell-cycle length than hASCs cultured using the conventional 2D method. This cell-cycle regulation is dependent on expression of cyclin D1. In summary, prior culturing of hASCs in a 3D microenvironment accelerates retroviral gene transduction by regulating cyclin D1 expression and accelerating cell-cycle synchronization. We conclude that priming via culturing in a 3D microenvironment facilitates efficient and rapid retroviral gene transduction of hASCs without inducing apoptosis.

Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming.

Primordial germ cells (PGCs) are reprogrammed into pluripotent embryonic germ cells (EGCs) under specific culture conditions, but the detailed mechanisms of PGC reprogramming have not yet been fully clarified. Previous studies have demonstrated that AKT, an important intracellular signaling molecule, promotes reprogramming of PGCs into EGCs. Because AKT likely inhibits p53 functions to enhance PGC reprogramming, and p53 negatively regulates cell cycle progression, we analyzed cell cycle changes in PGCs following AKT activation and found that the ratio of PGCs in the G1/G0 phase was decreased while that of PGCs in the G2/M phase was increased after AKT activation. We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1‐S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation. The results suggested that the characteristic cell cycle changes of PGCs by AKT activation are, at least in part, due to decreased expression of p27kip1 . We also investigated changes in histone H3K27 tri-methylation (H3K27me3) by AKT activation in PGCs, because we previously found that decreased H3K27me3 was involved in PGC reprogramming via upregulation of cyclin D1. We observed that AKT activation in PGCs resulted in H3K27 hypomethylation. In addition, DZNeP, an inhibitor of the H3K27 trimethyl transferase Ezh2, stimulated EGC formation. These results together suggested that AKT activation promotes G1-S transition and downregulates H3K27me3 to enhance PGC reprogramming.

Pax6 Lengthens G1 Phase and Decreases Oscillating Cdk6 Levels in Murine Embryonic Cortical Progenitors.

Pax6 is a key regulator of the rates of progenitor cell division in cerebral corticogenesis. Previous work has suggested that this action is mediated at least in part by regulation of the cell cycle gene Cdk6, which acts largely on the transition from G1 to S phase. We began the present study by investigating whether, in addition to Cdk6, other Pax6-regulated cell cycle genes are likely to be primary mediators of Pax6’s actions on cortical progenitor cell cycles. Following acute cortex-specific deletion of Pax6, Cdk6 showed changes in expression a day earlier than any other Pax6-regulated cell cycle gene suggesting that it is the primary mediator of Pax6’s actions. We then used flow cytometry to show that progenitors lacking Pax6 have a shortened G1 phase and that their Cdk6 levels are increased in all phases. We found that Cdk6 levels oscillate during the cell cycle, increasing from G1 to M phase. We propose a model in which loss of Pax6 shortens G1 phase by raising overall Cdk6 levels, thereby shortening the time taken for Cdk6 levels to cross a threshold triggering transition to S phase.