scholarly journals Myh6-driven Cre recombinase activates the DNA damage response and the cell cycle in the myocardium in the absence of loxP sites

2020 ◽  
Vol 13 (12) ◽  
pp. dmm046375
Author(s):  
Xinrui Wang ◽  
Amelia Lauth ◽  
Tina C. Wan ◽  
John W. Lough ◽  
John A. Auchampach
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Target Genes ◽  
Cre Recombinase ◽  
Eukaryotic Cell ◽  
Immunofluorescent Staining ◽  
Cardiovascular Research ◽  
Phosphohistone H3 ◽  
Damage Response

ABSTRACTRegeneration of muscle in the damaged myocardium is a major objective of cardiovascular research, for which purpose many investigators utilize mice containing transgenes encoding Cre recombinase to recombine loxP-flanked target genes. An unfortunate side effect of the Cre-loxP model is the propensity of Cre recombinase to inflict off-target DNA damage, which has been documented in various eukaryotic cell types including cardiomyocytes (CMs). In the heart, reported effects of Cre recombinase include contractile dysfunction, fibrosis, cellular infiltration and induction of the DNA damage response (DDR). During experiments on adult mice containing a widely used Myh6-merCremer transgene, the protein product of which is activated by tamoxifen, we observed large, transient, off-target effects of merCremer, some of which have not previously been reported. On Day 3 after the first of three daily tamoxifen injections, immunofluorescent microscopy of heart sections revealed that the presence of merCremer protein in myonuclei was nearly uniform, thereafter diminishing to near extinction by Day 6; during this time, cardiac function was depressed as determined by echocardiography. On Day 5, peaks of apoptosis and expression of DDR-regulatory genes were observed, highlighted by >25-fold increased expression of Brca1. Concomitantly, the expression of genes encoding cyclin-A2, cyclin-B2 and cyclin-dependent kinase 1, which regulate the G2/S cell-cycle transition, were dramatically increased (>50- to 100-fold). Importantly, immunofluorescent staining revealed that this was accompanied by peaks in Ki67, 5′-bromodeoxyuridine and phosphohistone H3 labeling in non-CMs, as well as CMs. We further document that tamoxifen-induced activation of merCremer exacerbates cardiac dysfunction following myocardial infarction. These findings, when considered in the context of previous reports, indicate that the presence of merCremer in the nucleus induces DNA damage and unscheduled cell-cycle activation. Although these effects are transient, the inclusion of appropriate controls, coupled with an awareness of the defects caused by Cre recombinase, are required to avoid misinterpreting results when using Cre-loxP models for cardiac regeneration studies.This article has an associated First Person interview with the first author of the paper.

scholarly journals Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina

2021 ◽  
Author(s):  
Swathy Babu ◽  
Yuki Takeuchi ◽  
Ichiro Masai
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Chromosome Segregation ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Damage Response ◽  
Dna Damage Responses

Btg3-associated nuclear protein (Banp) was originally identified as a nuclear matrix-associated protein and it functions as a tumor suppressor. At molecular level, Banp regulates transcription of metabolic genes via a CGCG-containing motif called the Banp motif. However, its physiological roles in embryonic development are unknown. Here we report that Banp is indispensable for DNA damage response and chromosome segregation during mitosis. Zebrafish banp mutants show mitotic arrest and apoptosis in developing retina. We found that DNA replication stress and tp53-dependent DNA damage responses were activated to induce apoptosis in banp mutants, suggesting that Banp is required for integrity of DNA replication and DNA damage repair. Furthermore, in banp mutants, chromosome segregation was not smoothly processed from prometaphase to anaphase, leading to a prolonged M-phase. Our RNA- and ATAC-sequencing identified 31 candidates for direct Banp target genes that carry the Banp motif. Interestingly, two chromosome segregation regulators, cenpt and ncapg, are included in this list. Thus, Banp directly regulates transcription of cenpt and ncapg to promote chromosome segregation during mitosis. Our findings provide the first in vivo evidence that Banp is required for cell-cycle progression and cell survival by regulating DNA damage responses and chromosome segregation during mitosis.

Abstract 400: Tip60 Depletion in Neonatal Cardiomyocytes Reduces the DNA Damage Response and Increases Cell-cycle Activation

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Xinrui Wang ◽  
Carri Lupton ◽  
Amelia Lauth ◽  
Tina C Wan ◽  
Michaela Patterson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Replicative Senescence ◽  
Phase Cell ◽  
Neonatal Cardiomyocytes ◽  
Cell Cycle Activation ◽  
Phosphohistone H3 ◽  
Damage Response ◽  
Cell Cycle Inhibitors

Tip60 ( T at i nteractive p rotein, 60 kD) is a tumor suppressor encoded by the Kat5 gene that functions as a pan-acetylase. Among its targets, Atm ( a taxia- t elangiectasia m utated) undergoes auto-phosphorylation (pATM) when acetylated by Tip60 to induce the DNA damage response (DDR). Because pAtm-induced activation of the DDR is known to cause proliferative senescence in neonatal cardiomyocytes (CMs), we hypothesize that Tip60 is one of the upstream components in this pathway. Using mice containing floxed Kat5 alleles and a tamoxifen-inducible Cre-recombinase transgene driven by Myh6 (Kat5 flox/flox;Myh6-merCremer ), we assessed the effect of depleting Tip60 from neonatal CMs via a single injection of tamoxifen on postnatal day 0 (P0). Immunofluorescent microscopy revealed reduced pAtm-positive CMs at three postnatal stages (P7, P12, P39), accompanied by activation of the cell-cycle as indicated by significantly increased percentages of 5’-bromodeoxyuridine (BrdU)-, phosphohistone H3 (pH3)- and Ki67-positive CMs. In addition to cell-cycle activation, Tip60 depletion promoted nuclear division followed by cytokinesis, as indicated by increased percentage of mononuclear diploid CMs at P12. Accordingly, qPCR revealed that Tip60 depletion increased expression of mRNAs encoding the fetal markers Nppa, Nppb, Myh7 , and Acta1 at P39, which in the absence of CM hypertrophy (assessed by WGA staining) suggested maintenance of a proliferation-competent state. These effects occurred concomitant with depressed levels of mRNAs encoding cell-cycle inhibitors ( Meis1, p27 ) and a trend toward increased levels of G 2 -phase cell-cycle activators ( cyclins A2, B1 ; Cdk1 ) in Tip60-depleted hearts. These findings suggest that depletion of Tip60 at birth inhibits the DDR and delays replicative senescence in neonatal CMs.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2

2019 ◽  
Vol 105 (3) ◽  
pp. 839-853
Author(s):  
Aglaia Kyrilli ◽  
David Gacquer ◽  
Vincent Detours ◽  
Anne Lefort ◽  
Frédéric Libert ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Iodide Uptake ◽  
Transcriptomic Response ◽  
Uptake Inhibition ◽  
Γ Radiation ◽  
Damage Response

Abstract Background The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

scholarly journals Differential DNA Damage Response of Peripheral Blood Lymphocyte Populations

2021 ◽  
Vol 12 ◽  
Author(s):  
Kerstin Felgentreff ◽  
Catharina Schuetz ◽  
Ulrich Baumann ◽  
Christian Klemann ◽  
Dorothee Viemann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ionizing Radiation ◽  
Dna Damage Response ◽  
Peripheral Blood ◽  
Low Dose ◽  
Lymphocyte Subsets ◽  
Mass Cytometry ◽  
Damage Response ◽  
Lymphocyte Populations

DNA damage occurs constantly in every cell triggered by endogenous processes of replication and metabolism, and external influences such as ionizing radiation and intercalating chemicals. Large sets of proteins are involved in sensing, stabilizing and repairing this damage including control of cell cycle and proliferation. Some of these factors are phosphorylated upon activation and can be used as biomarkers of DNA damage response (DDR) by flow and mass cytometry. Differential survival rates of lymphocyte subsets in response to DNA damage are well established, characterizing NK cells as most resistant and B cells as most sensitive to DNA damage. We investigated DDR to low dose gamma radiation (2Gy) in peripheral blood lymphocytes of 26 healthy donors and 3 patients with ataxia telangiectasia (AT) using mass cytometry. γH2AX, p-CHK2, p-ATM and p53 were analyzed as specific DDR biomarkers for functional readouts of DNA repair efficiency in combination with cell cycle and T, B and NK cell populations characterized by 20 surface markers. We identified significant differences in DDR among lymphocyte populations in healthy individuals. Whereas CD56+CD16+ NK cells showed a strong γH2AX response to low dose ionizing radiation, a reduced response rate could be observed in CD19+CD20+ B cells that was associated with reduced survival. Interestingly, γH2AX induction level correlated inversely with ATM-dependent p-CHK2 and p53 responses. Differential DDR could be further noticed in naïve compared to memory T and B cell subsets, characterized by reduced γH2AX, but increased p53 induction in naïve T cells. In contrast, DDR was abrogated in all lymphocyte populations of AT patients. Our results demonstrate differential DDR capacities in lymphocyte subsets that depend on maturation and correlate inversely with DNA damage-related survival. Importantly, DDR analysis of peripheral blood cells for diagnostic purposes should be stratified to lymphocyte subsets.

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