• DNA-damage
• Sca-1(+) progenitor
• Stem cell differentiation
• mESCs
• γH2AX

• Animals
• Mice
• Cell Differentiation/genetics
• DNA Breaks, Double-Stranded
• DNA Repair/genetics
• Embryonic Stem Cells
• Mouse Embryonic Stem Cells
• Histones/metabolism
• Histones/radiation effects

• Animals
• Mice
• S Phase/genetics
• Cell Cycle/genetics
• Gene Expression
• Mitosis
• Transcription Factors/genetics
• Genomic Instability
• DNA Replication/genetics

• S10 OD026929 NIH HHS
• R01 GM125089 NIGMS NIH HHS

• Aging
• Classic Galactosemia
• Eukaryotic initiation factor 2 alpha (eIF2ɑ)
• Follicle
• Granulosa cells
• Integrated stress
• Oocyte
• Ovary
• Primary ovarian insufficiency
• Translational control

• embryonic stem cells
• mitophagy

CTCF is a nuclear protein initially discovered for its role in enhancer-promoter insulation. It has been shown to play a role in genome architecture and in fact, its DNA binding sites are enriched at the borders of chromatin domains. Recently, we showed that depletion of CTCF impairs the DNA damage response to ionizing radiation. To investigate the relationship between chromatin domains and DNA damage repair, we present here clonogenic survival assays in different cell lines upon CTCF knockdown and ionizing irradiation. The application of a wide range of ionizing irradiation doses (0–10 Gy) allowed us to investigate the survival response through a biophysical model that accounts for the double-strand breaks’ probability distribution onto chromatin domains. We demonstrate that the radiosensitivity of different cell lines is increased upon lowering the amount of the architectural protein. Our model shows that the deficiency in the DNA repair ability is related to the changes in the size of chromatin domains that occur when different amounts of CTCF are present in the nucleus.

• CTCF
• DNA damage response
• DNA repair
• biophysical modeling
• cancer
• chromatin architecture
• chromatin domain
• clonogenic survival
• ionizing radiation
• radiosensitivity

• Chaperones
• Chromatin
• H2A.B
• H2A.X
• H2A.Z
• Histone variants
• Posttranslational modifications
• disease

• BAX
• DUX4
• Facioscapulohumeral muscular dystrophy
• Myotube
• Phospho(Ser139)-H2AX
• γH2AX

• DNA damage
• H2AX
• aging
• apoptosis
• cerebral cortex
• mitosis
• neurogenesis
• neurons
• subventricular zone

• Aging/metabolism
• Animals
• Brain/embryology
• Brain/metabolism
• Brain/pathology
• Embryo, Mammalian/metabolism
• Histones/metabolism
• Humans
• Oxidative Stress
• Phosphorylation

• Local funds form UniTO University of Turin

• Animals
• Benzhydryl Compounds
• Cell Differentiation
• Gene Expression Profiling
• Germ Cells
• Mice
• Phenols

• R01 ES027487 NIEHS NIH HHS

• chromatin
• epigenetics
• nuclear architecture
• phase separation
• pluripotency
• telomeres

• Wellcome Trust

• differentiation
• epigenetics
• histone variants
• pluripotent stem cells
• self-renewal

• BRG1
• G1
• H2A.X
• SNF2H
• SPT16
• TIP5
• cell cycle
• cell cycle arrest
• proliferation
• rDNA
• rRNA
• ribosomal biogenesis
• stem cells

Recognition of specific chromatin modifications by distinct structural domains within “reader” proteins plays a critical role in the maintenance of genomic stability. However, the specific mechanisms involved in this process remain unclear. Here we report that the PHD-Bromo tandem domain of tripartite motif-containing 66 (TRIM66) recognizes the unmodified H3R2-H3K4 and acetylated H3K56. The aberrant deletion of Trim66 results in severe DNA damage and genomic instability in embryonic stem cells (ESCs). Moreover, we find that the recognition of histone modification by TRIM66 is critical for DNA damage repair (DDR) in ESCs. TRIM66 recruits Sirt6 to deacetylate H3K56ac, negatively regulating the level of H3K56ac and facilitating the initiation of DDR. Importantly, Trim66-deficient blastocysts also exhibit higher levels of H3K56ac and DNA damage. Collectively, the present findings indicate the vital role of TRIM66 in DDR in ESCs, establishing the relationship between histone readers and maintenance of genomic stability.

TRIM66 protein has an N-terminal tripartite motif and a C-terminal PHD Bromodomain. Here the authors show the specific histone modification recognition of TRIM66-PHD-Bromodomain through crystallography and biochemistry assay, and further reveal that TRIM66 recognition of certain histone modification is important for DNA damage repair in ESCs.

Many studies have reported that human-induced pluripotent stem (hiPS)/embryonic stem (hES) cells have an exceptional ability to repair damaged DNA. Moreover, unlike differentiated cells, hES cells have features and mechanisms such as apoptosis-prone mitochondria, which prevent any changes in genetic information caused by DNA damage to be transmitted to their descendants. Type-A (dark) spermatogonia and cancer stem cells are thought to be dormant. However, hiPS/hES cells, the so-called stem cells used in regenerative medicine, generally have a high proliferative capacity. This suggests that in these cells, oxidative DNA damage associated with vigorous proliferation and DNA scission associated with replication occur frequently. Although pluripotency according to change of genomic structure is well studied, the change of DNA repair through reprogramming has not been well studied.

We analyzed the expression of DNA repair-related genes in hiPS cells using microarray and western blotting analyses and assessed changes in PARP activity through reprogramming.

Through reprogramming, hiPS cells were found to upregulate poly (ADP-ribose) polymerase (PARP) activity and genes regulating homologous recombination (HR). Simultaneously, the expression level of genes involved in non-homologous end joining (NHEJ) was not high, suggesting that at least at the gene expression level, frequently occurring DNA scission is preferentially dealt with via HR instead of NHEJ. Also, reflecting the high proliferative activity, genes related to mismatch repair (MMR) were upregulated through reprogramming. Conversely, error-prone polymerase was downregulated through reprogramming. These are also likely to be the mechanisms preventing changes in genetic information.

High PARP activity and HR-related gene expression in hiPS cells were achieved through reprogramming and likely facilitate precise genome editing in these cells in exchange for a high possibility of cell death.

• BLM
• BLM, the gene defective in Bloom's syndrome
• DNA repair
• HDF, human dermal fibroblast
• Homologous recombination
• MSC, mesenchymal stromal cell
• PARP
• PARP, poly (ADP-ribose) polymerase
• RAD51
• ROS, Reactive Oxygen Species
• Reprogramming
• iPS, induced pluripotent stem

Research in the stem cell field has traditionally focused on understanding key transcriptional factors that provide pluripotent cell identity. However, much less is known about other critical non-transcriptional signaling networks that govern stem cell identity. Although we continue to gain critical insights into the mechanisms underlying mitochondrial morphology and function during cellular reprogramming – the process of reverting the fate of a differentiated cell into a stem cell, many uncertainties remain. Recent studies suggest an emerging landscape in which mitochondrial morphology and function have an active role in maintaining and regulating changes in cell identity. In this review, we will focus on these emerging concepts as crucial modulators of cellular reprogramming. Recognition of the widespread applicability of these concepts will increase our understanding of the mitochondrial mechanisms involved in cell identity, cell fate and disease.

• BCL-2 family
• apoptosis
• mitochondria
• mitochondrial dynamics

• Caenorabditis elegans
• DNA damage
• DNA damage tolerance
• Drosophila melanogaster
• Xenopus laevis
• iPSCs
• mouse embryonic stem cells
• replication stress
• zebrafish

• Animals
• Cell Cycle/genetics
• DNA/biosynthesis
• DNA/genetics
• DNA Damage/genetics
• DNA Replication/genetics
• Embryonic Development/genetics
• Genome/genetics
• Genomic Instability/genetics
• Humans
• S Phase/genetics

• R01 CA174966 NCI NIH HHS
• U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)

• DNA damage response
• cellular reprogramming
• chromatin remodeling
• induced pluripotent stem cells

• Animals
• Cell Differentiation
• Cells, Cultured
• Cellular Reprogramming
• Epigenesis, Genetic
• Gene Expression Profiling
• HEK293 Cells
• Humans
• Kruppel-Like Factor 4
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/metabolism
• Transcription Factors

• R01 GM105772 NIGMS NIH HHS
• R01 GM107092 NIGMS NIH HHS
• S10 OD030363 NIH HHS
• S10 RR027990 NCRR NIH HHS
• S10 OD018521 NIH HHS
• DP2 GM123507 NIGMS NIH HHS

• DNA damage
• DNA replication
• Induced pluripotent stem cells
• Mesenchymal stromal cells
• γH2AX

Early mouse development is regulated and accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2). Previously, we provided insights into its role in post-implantation development (Zylicz et al., 2015). Here we explore the impact of depleting the maternally inherited G9a in oocytes on development shortly after fertilisation. We show that G9a accumulates typically at 4 to 8 cell stage to promote timely repression of a subset of 4 cell stage-specific genes. Loss of maternal inheritance of G9a disrupts the gene regulatory network resulting in developmental delay and destabilisation of inner cell mass lineages by the late blastocyst stage. Our results indicate a vital role of this maternally inherited epigenetic regulator in creating conducive conditions for developmental progression and on cell fate choices.

• blastocyst
• chromosomes
• developmental biology
• embryogenesis
• epigenetics
• gene expression
• mouse
• stem cells
• transcription

• embryonic stem cells
• genome stability
• homologous recombination
• replication stress

• DNA damage sensitivity
• DNA repair
• Embryonic stem cells
• Ronin/Thap11

• DNA damage
• DNA methylation
• epigenetics
• germ cells
• heterochromatin

Despite the well-demonstrated efficacy of stem cell (SC) therapy, this approach has a number of key drawbacks. One important concern is the response of pluripotent SCs to treatment with ionizing radiation (IR), given that SCs used in regenerative medicine will eventually be exposed to IR for diagnostic or treatment-associated purposes. Therefore, the aim of the present study was to examine and compare early IR-induced responses of pluripotent SCs to assess their radioresistance and radiosensitivity. In the present study, 3 cell lines; human embryonic SCs (hESCs), human induced pluripotent SCs (hiPSCs) and primary human dermal fibroblasts (PHDFs); were exposed to IR at doses ranging from 0 to 15 gray (Gy). Double strand breaks (DSBs), and the gene expression of the following DNA repair genes were analyzed: P53; RAD51; BRCA2; PRKDC; and XRCC4. hiPSCs demonstrated greater radioresistance, as fewer DSBs were identified, compared with hESCs. Both pluripotent SC lines exhibited distinct gene expression profiles in the most common DNA repair genes that are involved in homologous recombination, non-homologous end-joining and enhanced DNA damage response following IR exposure. Although hESCs and hiPSCs are equivalent in terms of capacity for pluripotency and differentiation into 3 germ layers, the results of the present study indicate that these 2 types of SCs differ in gene expression following exposure to IR. Consequently, further research is required to determine whether hiPSCs and hESCs are equally safe for application in clinical practice. The present study contributes to a greater understanding of DNA damage response (DDR) mechanisms activated in pluripotent SCs and may aid in the future development of safe SC-based clinical protocols.

Early embryonic cleavages are characterized by short and highly synchronous cell cycles made of alternating S- and M-phases with virtually absent gap phases. In this contracted cell cycle, the duration of DNA synthesis can be extraordinarily short. Depending on the organism, the whole genome of an embryo is replicated at a speed that is between 20 to 60 times faster than that of a somatic cell. Because transcription in the early embryo is repressed, DNA synthesis relies on a large stockpile of maternally supplied proteins stored in the egg representing most, if not all, cellular genes. In addition, in early embryonic cell cycles, both replication and DNA damage checkpoints are inefficient. In this article, we will review current knowledge on how DNA synthesis is regulated in early embryos and discuss possible consequences of replicating chromosomes with little or no quality control.

• Drosophila
• S-phase
• Xenopus
• cell cycle
• checkpoint
• development
• translesion synthesis

The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E.

• Cell cycle
• ESCs
• MEFs
• Pax7
• differentiation
• mouse

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, believed to be the driving factor in the pathogenesis of this MM subtype. MMSET overexpression in MM leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3), as well as changes in proliferation, gene expression, and chromatin accessibility. Prior work linked methylation of histones to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET may play a role in DNA damage repair and response. In U2OS cells, we found that MMSET is required for efficient non-homologous end joining as well as homologous recombination. Loss of MMSET led to loss of expression of several DNA repair proteins, as well as decreased recruitment of DNA repair proteins to sites of DNA double strand breaks (DSBs). Using genetically matched MM cell lines that had either high (pathological) or low (physiological) expression of MMSET, we found that MMSET high cells had increased damage at baseline. Upon addition of a DNA damaging agent, MMSET high cells repaired DNA damage at an enhanced rate and continued to proliferate, whereas MMSET low cells accumulated DNA damage and entered cell cycle arrest. In a murine xenograft model using t(4;14)+ KMS11 MM cells harboring an inducible MMSET shRNA, depletion of MMSET enhanced the efficacy of chemotherapy, inhibiting tumor growth and extending survival. These findings help explain the poorer prognosis of t(4;14) MM and further validate MMSET as a potential therapeutic target in MM and other cancers.

• ChIP-seq, Chromatin Immunoprecipitation Sequencing
• EB, embryoid body
• EHP cells, endoderm/hepatic progenitor cells
• ESCs, embryonic stem cells
• MEFs, mouse embryonic fibroblasts
• NLBs, nucleolar-like bodies
• PcG, Polycomb group
• TSS, transcription start site
• chromatin dynamics
• embryonic stem cell
• histone variant
• iPSCs, induced pluripotent stem cells
• pluripotency
• self-renewal

Normal stem cells from tissues often exhibiting radiation injury are highly radiosensitive and exhibit a muted DNA damage response, in contrast to differentiated progeny. These radioresponses can be attributed to unique epigenetic regulation in stem cells, identifying potential therapeutic targets for radioprotection.

Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non–stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation–induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylation (γH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment.

In many animals, primordial germ cells (PGCs) migrate into developing gonads. There, they proliferate and differentiate into female and male germ stem cells (GSCs), oogonia and spermatogonia, respectively. Few studies have focused on the molecular mechanisms underlying the development of GSC sex determination. Here, we investigated the expression of the transcription factor Dmrt1 and a phosphorylated form of the histone variant H2AX (γH2AX) during gonadal development in Xenopus laevis. During early sexual differentiation, Dmrt1 was expressed in the GSCs of the ZW (female) and ZZ (male) gonads as well as somatic cells of the ZZ gonads. Notably, the PGCs and primary GSCs contained large, unstructured nuclei, whereas condensed, rounder nuclei appeared only in primary oogonia during tadpole development. After metamorphosis, Dmrt1 showed its expression in secondary spermatogonia, but not in secondary oogonia. Like Dmrt1, γH2AX was expressed in the nuclei of primary GSCs in early developing gonads. However, after metamorphosis, γH2AX expression continued in primary and secondary spermatogonia, but was barely detected in the condensed nuclei of primary oogonia. Taken together, these observations indicate that spermatogonia tend to retain PGC characteristics, compared to oogonia, which undergo substantial changes during gonadal differentiation in X. laevis. Our findings suggest that Dmrt1 and γH2AX may contribute to the maintenance of stem cell identity by controlling gene expression and epigenetic changes, respectively.

• Xenopus laevis
• germ cells
• oogonia
• sexual dimorphism
• spermatogonia
• transcription factor

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.

In fast proliferating embryonic stem cells (ESC) the DNA damage response is activated by mechanisms that are as yet elusive. Here, Ahuja et al. link the DNA damage response to replication stress in mouse ESCs, caused by a short G1 phase, and propose fork remodelling as maintaining genome stability in embryos.

Regenerative medicine is a rapidly growing field that holds promise for the treatment of many currently unresponsive diseases. Stem cells (SCs) are undifferentiated cells with long-term self-renewal potential and the capacity to develop into specialized cells. SC-based therapies constitute a novel and promising concept in regenerative medicine. Radiotherapy is the most frequently used method in the adjuvant treatment of tumorous alterations. In the future, the usage of SCs in regenerative medicine will be affected by their regular and inevitable exposure to ionizing radiation (IR). This phenomenon will be observed during treatment as well as diagnosis. The issue of the genetic stability of SCs and cells differentiated from SCs is crucial in the context of the application of these cells in clinical practice. This review examines current knowledge concerning the DNA repair mechanisms (base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining) of SCs in response to the harmful effects of genotoxic agents such as IR and chemotherapeutics.

• DNA damage response
• DNA repair
• chemotherapeutics
• genetic stability
• ionizing radiation
• pluripotent stem cells

• DNA damage
• Differentiation
• Embryonic stem cells
• Histone demethylase
• Histone methylation
• Polycomb

• apoptosis
• dicer
• embryonic stem cells
• medulloblastoma
• p53
• replicative stress

The identification of cancer stem cells (CSCs) that are responsible for tumor initiation, growth, metastasis, and therapeutic resistance might lead to a new thinking on cancer treatments. Similar to stem cells, CSCs also display high resistance to radiotherapy and chemotherapy with genotoxic agents. Thus, conventional therapy may shrink the tumor volume but cannot eliminate cancer. Eradiation of CSCs represents a novel therapeutic strategy. CSCs possess a highly efficient DNA damage response (DDR) system, which is considered as a contributor to the resistance of these cells from exposures to DNA damaging agents. Targeting of enhanced DDR in CSCs is thus proposed to facilitate the eradication of CSCs by conventional therapeutics. To achieve this aim, a better understanding of the cellular responses to DNA damage in CSCs is needed. In addition to the protein kinases and enzymes that are involved in DDR, other processes that affect the DDR including chromatin remodeling should also be explored.

• Cancer stem cell
• DNA damage response
• DNA repair
• Cancer therapy

• R01 CA151248 NCI NIH HHS

• Cell Differentiation
• DNA Breaks, Double-Stranded/drug effects
• DNA Damage/genetics
• Humans
• Mesenchymal Stem Cells/metabolism
• Oxidative Stress

• cell fate
• chromatin
• induced pluripotent stem cells
• reprogramming
• transcription factors

In the last decade, many papers highlighted that the histone variant H2AX and its phosphorylation on Ser 139 (γH2AX) cannot be simply considered a specific DNA double-strand-break (DSB) marker with a role restricted to the DNA damage response, but rather as a ‘protagonist’ in different scenarios. This review will present and discuss an up-to-date view regarding the ‘non-canonical’ H2AX roles, focusing in particular on possible functional and structural parts in contexts different from the canonical DNA DSB response. We will present aspects concerning sex chromosome inactivation in male germ cells, X inactivation in female somatic cells and mitosis, but will also focus on the more recent studies regarding embryonic and neural stem cell development, asymmetric sister chromosome segregation in stem cells and cellular senescence maintenance. We will discuss whether in these new contexts there might be a relation with the canonical DNA DSB signalling function that could justify γH2AX formation. The authors will emphasize that, just as H2AX phosphorylation signals chromatin alteration and serves the canonical function of recruiting DSB repair factors, so the modification of H2AX in contexts other than the DNA damage response may contribute towards creating a specific chromatin structure frame allowing ‘non-canonical’ functions to be carried out in different cell types.

Embryonic stem cells (ESCs) enable rapid proliferation that also causes DNA damage. To maintain genomic stabilization during rapid proliferation, ESCs must have an efficient system to repress genotoxic stress. Here, we show that withdrawal of leukemia inhibitory factor (LIF), which maintains the self-renewal capability of mouse ESCs (mESCs), significantly inhibits the cell proliferation and DNA damage of mESCs and upregulates the expression of miR-590. miR-590 promotes single-strand break (SSB) and double-strand break (DSB) damage repair, thus slowing proliferation of mESCs without influencing stemness. miR-590 directly targets Activin receptor type 2a (Acvr2a) to mediate Activin signaling. We identified the homologous recombination-mediated repair (HRR) gene, Rad51b, as a downstream molecule of the miR-590/Acvr2a pathway regulating the SSB and DSB damage repair and cell cycle. Our study shows that a miR-590/Acvr2a/Rad51b signaling axis ensures the stabilization of mESCs by balancing DNA damage repair and rapid proliferation during self-renewal.

•

miR-590 promotes DNA damage repair and slows proliferation by targeting Acvr2a