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Mesenchymal stem cells (MSCs) are multilineage adult stem cells with considerable potential for cell-based regenerative therapies. In vitro expansion changes their epigenetic and cellular properties, with a poorly understood impact on DNA damage response (DDR) and genome stability. We report here results of a transcriptome-based pathway analysis of in vitro-expanded human bone marrow-derived mesenchymal stem cell (hBM-MSCs), supplemented with cellular assays focusing on DNA double-strand break (DSB) repair. Gene pathways affected by in vitro aging were mapped using gene ontology, KEGG, and GSEA, and were found to involve DNA repair, homologous recombination (HR), cell cycle control, and chromosomal replication. Assays for the recognition (γ-H2AX + 53BP1 foci) and repair (pBRCA1 + γ-H2AX foci) of X-ray-induced DNA DSBs in hBM-MSCs show that over a period of 8 weeks of in vitro aging (i.e., about 10 doubling times), cells exhibit a reduced DDR and a higher fraction of residual DNA damage. Furthermore, a distinct subpopulation of cells with impaired DNA DSB recognition was observed. Several genes that participate in DNA repair by HR (e.g., Rad51, Rad54, BRCA1) show a 2.3- to fourfold reduction of their mRNA expression by qRT-PCR. We conclude that the in vitro expansion of hMSCs can lead to aging-related impairment of the recognition and repair of DNA breaks., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G 1 -like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

PC4 is an abundant single-strand DNA binding protein that has been implicated in transcription and DNA repair. Here, we show that PC4 is involved in the cellular DNA damage response. To elucidate the role, we used the DT40 chicken B cell model, which produces clustered DNA lesions at Ig loci via the action of activation-induced deaminase. Our results help resolve key aspects of immunoglobulin diversification and suggest an essential role of PC4 in repair pathway choice. We show that PC4 ablation in gene conversion (GC)-active cells significantly disrupts GC but has little to no effect on targeted homologous recombination. In agreement, the global double-strand break repair response, as measured by γH2AX foci analysis, is unperturbed 16 hours post irradiation. In cells with the pseudo-genes removed (GC inactive), PC4 ablation reduced the overall mutation rate while simultaneously increasing the transversion mutation ratio. By tagging the N-terminus of PC4, gene conversion and somatic hypermutation are all but abolished even when native non-tagged PC4 is present, indicating a dominant negative effect. Our data point to a very early and deterministic role for PC4 in DNA repair pathway re-routing.

Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(Δ/Δ) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of γH2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1(-/-) animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated A(aligned) (A(al)) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1(-/-) gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1(-/-) testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

Meiosis is central to the formation of haploid gametes or spores in that it segregates homologous chromosomes and halves the chromosome number. A prerequisite of this genome bisection is the pairing of homologous chromosomes during the first meiotic prophase. When budding yeast cells are induced to undergo meiosis, this has profound consequences for nuclear structure: after premeiotic DNA replication centromeres disperse, while telomeres move about the nuclear periphery and temporarily cluster during the leptotene/zygotene transition (bouquet stage) of the prophase to first meiotic division. In vegetative cells, Hdf1p (yKu) and the myosin-like proteins Mlp1p and Mlp2p have been suggested to contribute to the organization of silent chromatin, tethering of telomeres to the nuclear periphery, DNA repair, and telomere maintenance. Here, we investigated by molecular cytology whether yKu and Mlp proteins contribute to telomere and chromosome dynamics in meiosis. It was found that mlp1 Delta mlp2 Delta double-mutant cells undergo centromere dispersion, telomere clustering, homologue pairing, and sporulation like wild type. On the other hand, cells deficient for yKu underwent meiosis-specific chromosomal events with a delay, while they eventually sporulated like wild type. These results suggest that the absence of yKu not only affects vegetative nuclear architecture (Laroche et al., 1998) but also interferes with the ordered occurrence of chromosome dynamics during first meiotic prophase.

The entry into meiosis is characterized by a lengthy premeiotic S phase and a reorganization of the nuclear architecture. Analysis of centromere and telomere dynamics in wild-type Saccharomyces cerevisiae meiosis suggests that resolution of vegetative centromere and telomere clusters are independent events differently connected to premeiotic S phase. Absence of the B-type cyclin Clb5 or the Set1 histone methyltransferase leads to a delay of premeiotic S phase by separate mechanisms. In clb5Delta cells, centromere cluster resolution appears normal, whereas dissolution of the vegetative telomere clusters is impaired and meiosis-specific clustering of telomeres, i.e. bouquet formation, is grossly delayed. In set1Delta cells, centromere and telomere redistribution are both impaired and bouquet nuclei are absent, despite proper location of the meiosis-specific telomere protein Ndj1. Thus, centromere and telomere redistribution at the onset of prophase I is differentially regulated, with centromere dispersion occurring independently of premeiotic S phase. The normal kinetics of dissolution of the vegetative telomere clusters in a set1Delta mec1-1 mutant suggests the presence of a checkpoint that limits the dispersion of telomeres in absence of Set1.

The myogenic regulatory factors, Mrf4 and Myf5, play a key role in skeletal muscle formation. An enhancer trap approach, devised to isolate positive-acting elements from a 200-kb YAC covering the mouse Mrf4-Myf5 locus in a C2 myoblast assay, yielded an enhancer, A17, which mapped at -8 kb 5' of Mrf4 and -17 kb 5' of Myf5. An E-box bound by complexes containing the USF transcription factor is critical for enhancer activity. In transgenic mice, A17 gave two distinct and mutually exclusive expression profiles before birth, which correspond to two phases of Mrf4 transcription. Linked to the Tk or Mrf4 minimal promoters, the nlacZ reporter was expressed either in embryonic myotomes, or later in fetal muscle, with the majority of Mrf4 lines showing embryonic expression. When linked to the Myf5 minimal promoter, only fetal muscle expression was detected. These observations identify A17 as a sequence that targets sites of myogenesis in vivo and raise questions about the mutually exclusive modes of expression and possible promoter/enhancer interactions at the Mrf4-Myf5 locus.

Previous studies have shown that the RAD51 and DMC1 genes of Saccharomyces cerevisiae encode homologs of the Escherichia coli RecA strand exchange enzyme. Results presented here demonstrate that the dmc1 and rad51 mutants undergo nearly complete chromosome synapsis, but synaptonemal complex formation is delayed substantially compared with wild type. In the zip1 mutant, chromosomes are paired homologously, but not synapsed, and the protein backbones (axial elements) of each pair of chromosomes are connected intimately to each other at a few sites referred to herein as axial associations. dmc1 zip1 and rad51 zip1 double mutants assemble axial elements that are not obviously associated, demonstrating that the Dmc1 and Rad51 proteins are required to establish or stabilize axial associations. We propose that axial associations serve to promote meiotic chromosome synapsis and that the absence of these associations accounts for the delayed and inefficient synapsis observed in dmc1 and rad51 strains. During meiosis in haploid yeast, chromosome synapsis takes place between nonhomologous chromosome segments. In a zip1 haploid, axial associations are not apparent, suggesting that these associations depend on interactions between homologous sequences.

Nuclear factor I (NFI) is a member of a family of dimeric DNA-binding proteins that are involved both in the initiation of adenovirus DNA replication and in the stimulation of transcriptional activation. We have used fluorescence in situ hybridisation (FISH) to map one of four known genes encoding an NFI protein, the human NFI/X gene, to chromosome 19p1.3. Secondary sites of hybridisation observed at 5p1.5, 1q4.2-4.4, 1p2.1-2.2, and 20p1.3 most likely are attributable to partial sequence homologies with related NFI genes.