Recent studies suggest that transgenerational genome instability may be epigenetic in nature and mediated via altered DNA methylation and microRNAome. Here, we investigated the nature and mechanisms underlying the disruption of DNA methylation and microRNA expression status in the germline and progeny of exposed parents. We have found that paternal irradiation leads to upregulation of the miR-29 family in the exposed male germline, which causes decreased expression of de novo methyltransferase, DNA methyltransferase 3a, and profound hypomethylation of long interspersed nuclear elements 1 (LINE1) and short interspersed nuclear elements B2 (SINE B2). Epigenetic changes in the male germline further resulted in deleterious effects in the somatic thymus tissue from the progeny of exposed animals, including hypomethylation of LINE1 and SINE B2. Hypomethylation of LINE1 and SINE B2 in the thymus tissue from the progeny was associated with a significant decrease in the levels of lymphoid-specific helicase (LSH) that is crucial for the maintenance of methylation and silencing of repetitive elements. Furthermore, we noted a significant upregulation of miR-468 that targets LSH and leads to its decreased expression in thymus in the progeny of exposed parents. We suggest that miR-468-mediated suppression of LSH leads to aberrant methylation of LINE1 and SINE B2. In summary, altered microRNAome and hypomethylation of retroelements constitute deleterious effects that may significantly influence genome stability of the parental germline and consequently cause genome instability in the progeny.

It is now well accepted that parental whole body irradiation causes transgenerational genome and epigenome instability in the offspring. The majority of human exposures to radiation, such as therapeutic and diagnostic irradiation, are localized and focused. The potential of localized body-part exposures to affect the germline and thus induce deleterious changes in the progeny has not been studied. To investigate whether or not the paternal cranial irradiation can exert deleterious changes in the protected germline, we studied the accumulation of DNA damage in the shielded testes tissue. Here we report that the localized paternal cranial irradiation results in a significant accumulation of unrepaired DNA lesions in sperm cells and leads to a profound epigenetic dysregulation in the unexposed progeny conceived a week after paternal exposure.

MicroRNAs as potent regulators of gene expression are involved in spermatogenesis, yet their role in response of germline to genotoxic stress is obscure. We studied the microRNAome profile of X-ray irradiated mouse testes using the microarray technique. We found that radiation exposure significantly affected microRNA expression in testes. Mir-709 was the most abundant in both control and irradiated testes, and a big difference in miR-709 levels was observed between the control and exposed group. We found that miR-709 targets the Brother of the Regulator of Imprinted Sites (BORIS), an important regulator of DNA methylation and imprinting. Here, we for the first time show that the DNA damageinduced and ATR/Rfx1-mediated increase of miR-709 expression in exposed testes may be a protective mechanism that effectively decreases a cellular level of BORIS to prevent massive aberrant erasure of DNA methylation after radiation exposure.

Ionizing radiation (IR) is a curative treatment for many human malignancies, an important diagnostic modality, and a pivotal preparative regimen for bone marrow transplantation. On the other hand, IR is a potent damaging agent that can affect a variety of processes in directly exposed cells, in their descendents, and in neighboring un-irradiated naïve 'bystander' cells. Accumulation of DNA damage caused by IR in conjunction with disrupted cellular regulation processes can lead to genome instability in the germline, and therefore to transgenerational genome instability in offspring of exposed males. The exact mechanisms of IR-induced genome instability in directly exposed and in naïve bystander germ cells remain obscure, yet accumulating evidence points to the role of DNA damage and DNA methylation changes in genome instability development. In the current study, we used a well-established murine model to define the role of DNA methylation, DNA damage, as well as two important germline regulators of DNA methylation, Brother of the Regulator of Imprinted Sites (BORIS) and CCCTC binding factor (CTCF), in IR and bystander responses of the male germline. Here we report that irradiation leads to a significant accumulation of DNA damage in the exposed and bystander testis tissue, and to altered DNA methylation and dysregulated BORIS expression in the exposed testis tissue. The possible molecular mechanisms and biological consequences of the observed changes are discussed.

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