The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation.

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.

Adipose Tissue Transcriptome Is Related to Pollutant Exposure in Polar Bear Mother-Cub Pairs from Svalbard, Norway.

Being at the food chain apex, polar bears (Ursus maritimus) are highly contaminated with persistent organic pollutants (POPs). Females transfer POPs to their offspring through gestation and lactation; therefore, young cubs present higher POPs concentrations than their mothers. Recent studies suggest that POPs affect the lipid metabolism in female polar bears; however, the mechanisms and impact on their offspring remain unknown. Here, we hypothesized that exposure to POPs differentially alters genome-wide gene transcription in the adipose tissue from mother polar bears and their cubs, highlighting molecular differences in response between adults and young. Adipose tissue biopsies were collected from 13 adult female polar bears and their twin cubs in Svalbard, Norway, in April 2011, 2012, and 2013. Total RNA extracted from biopsies was subjected to next-generation RNA sequencing. Plasma concentrations of summed polychlorinated biphenyls, organochlorine pesticides, and polybrominated diphenyl ethers in mothers ranged from 897 to 13620 ng/g wet weight and were associated with altered adipose tissue gene expression in both mothers and cubs. In mothers, 2502 and 2586 genes in total were positively and negatively, respectively, correlated to POP exposure, whereas in cubs, 2585 positively and 1690 negatively genes. Between mothers and cubs, 743 positively and negatively genes overlapped between mothers and cubs suggesting partially shared molecular responses to ΣPOPs. ΣPOP-associated genes were involved in numerous metabolic pathways in mothers and cubs, indicating that POP exposure alters the energy metabolism, which, in turn, may be linked to metabolic dysfunction.

Histomorphologic Analysis of the Late-term Rat Fetus and Placenta.

Histological examination of the rat placenta and fetus is uncommon. Toxicological studies mainly rely on gross examination of the fetus and on fetal and placental weights. These are often insufficient to assess the fetal and placental toxicity of xenobiotics. The small size of the fetus makes its dissection labor-intensive. Thus, our objective was to develop a simple and accurate technique to evaluate the rat fetus and placenta. Sprague-Dawley rat fetuses at gestational day 19.5 ( n = 18) and their placentas ( n = 32) were fixed in formalin. Placentas were cut transversally in the center. Fetuses were cut following a freehand whole-body serial sectioning diagram adapted from Wilson's method. Sections were stained with hematoxylin-eosin-phloxine-saffron, and histomorphometry was used to measure the area of the fetal placental region (27.2 ± 1.7 mm2), including the labyrinth (22.2 ± 1.0 mm2) and the basal zone (4.8 ± 0.8 mm2). Our whole-fetus serial sectioning technique resulted in 12 precise cutting planes that fit on 3 histological slides, enabling the examination of most organs without labor-intensive dissection. Quantitative analysis of placental areas improves the understanding of the pathogenesis of treatment-related changes. This technique provides a standardized method for future research in pertinent fields such as developmental biology and toxicology.

Exposure to environmental contaminants and folic acid supplementation intergenerationally impact fetal skeleton development through the paternal lineage in a rat model.

Persistent organic pollutants (POPs) are ubiquitous in the environment, which is of concern since they are broadly toxic for wildlife and human health. It is generally accepted that maternal prenatal folic acid supplementation (FA) may beneficially impact offspring development, but it has been recently shown that the father's exposures also influence the health of his offspring. Bone is an endocrine organ essential for whole-body homeostasis and is susceptible to toxicants. Herein, we tested the hypotheses that prenatal paternal exposure to POPs induces developmental bone disorders in fetuses across multiple generations and that FA supplementation attenuates these disorders. We used a four-generation rat model, in which F0 founder females were divided into four treatment groups. F0 females were gavaged with corn oil or an environmentally-relevant POPs mixture and fed either a control diet (2 mg FA/kg), or FA supplemented diet (6 mg FA/kg) before mating and until parturition (four treatments in total). After the birth of the F1 litters, all F0 females and subsequent generations received the FA control diet. Staining with alcian blue and alizarin red S of male and female fetal skeletons was performed at Gestational Day 19.5. Paternal direct and ancestral exposure to POPs delayed bone ossification and decreased the length of long limb bones in fetuses. Maternal FA supplementation did not counteract the POPs-associated delayed fetal ossification and reduced long bone length. In conclusion, prenatal paternal POPs exposure causes developmental bone abnormalities over multiple generations, which were not corrected by maternal FA supplementation.

Beyond fertilisation: How the paternal environment influences future generations.

In light of the relatively ignored role of paternal influences on offspring development and increasing societal concerns regarding possible health consequences of chemical exposures, our team has addressed the overall hypothesis that environmentally-relevant levels of contaminants have long-lasting effects that are transmitted through the paternal lineage. This review focuses on our research examining the impact of developmental exposure to toxicants and nutrients on the phenotype and epigenome of the male and of his subsequent generations. This report is intended to encourage animal andrologists as well as the domestic animal production industry to increase their consideration of the sire's environment in the context of agricultural productivity.

Maternal folic acid supplementation does not counteract the deleterious impact of prenatal exposure to environmental pollutants on lipid homeostasis in male rat descendants.

Prenatal exposure to persistent organic pollutants (POPs) has been associated with the development of metabolic syndrome-related diseases in offspring. According to epidemiological studies, father's transmission of environmental effects in addition to mother's can influence offspring health. Moreover, maternal prenatal dietary folic acid (FA) may beneficially impact offspring health. The objective is to investigate whether prenatal FA supplementation can overcome the deleterious effects of prenatal exposure to POPs on lipid homeostasis and inflammation in three generations of male rat descendants through the paternal lineage. Female Sprague-Dawley rats (F0) were exposed to a POPs mixture (or corn oil) +/- FA supplementation for 9 weeks before and during gestation. F1 and F2 males were mated with untreated females. Plasma and hepatic lipids were measured in F1, F2, and F3 males after 12-h fast. Gene expression of inflammatory cytokines was determined by qPCR in epididymal adipose tissue. In F1 males, prenatal POPs exposure increased plasma lipids at 14 weeks old and hepatic lipids at 28 weeks old and prenatal FA supplementation decreased plasma total cholesterol at 14 weeks old. Prenatal POPs exposure decreased plasma triglycerides at 14 weeks old in F2 males. No change was observed in inflammatory markers. Our results show an impact of the paternal lineage on lipid homeostasis in rats up to the F2 male generation. FA supplementation of the F0 diet, regardless of POPs exposure, lowered plasma cholesterol in F1 males but failed to attenuate the deleterious effects of prenatal POPs exposure on plasma and hepatic lipids in F1 males.

Prenatal Exposure to Environmentally-Relevant Contaminants Perturbs Male Reproductive Parameters Across Multiple Generations that are Partially Protected by Folic Acid Supplementation.

The paternal environment is thought to influence sperm quality and future progeny may also be impacted. We hypothesized that prenatal exposure to environmentally-relevant contaminants impairs male reproduction, altering embryo gene expression over multiple generations. Folic acid (FA) can improve sperm quality and pregnancy outcomes, thus we further hypothesized that FA mitigates the contaminants. Sprague-Dawley F0 female rats treated with persistent organic pollutants (POPs) or corn oil and fed basal or supplemented FA diets, then used to yield four generations of litters. Only F0 females received POPs and/or FA treatments. In utero POPs exposure altered sperm parameters in F1, which were partly rescued by FA supplementation. Paternal exposure to POPs reduced sperm quality in F2 males, and the fertility of F3 males was modified by both POPs and FA. Ancestral FA supplementation improved sperm parameters of F4 males, while the POPs effect diminished. Intriguingly, F3 males had the poorest pregnancy outcomes and generated the embryos with the most significantly differentially expressed genes. Early-life exposure to POPs harms male reproduction across multiple generations. FA supplementation partly mitigated the impact of POPs. The two-cell embryo transcriptome is susceptible to paternal environment and could be the foundation for later pregnancy outcomes.

A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells.

Conventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small- and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships.

The contribution of the major metabolite 4'-O-methylmonoHER to the antioxidant activity of the flavonoid monoHER.

The antioxidant flavonoid 7-mono-O-(ß-hydroxyethyl)-rutoside (monoHER) effectively protects against doxorubicin-induced cardiotoxicity in mice. Doxorubicin is a very effective anticancer drug. The clinical use of doxorubicin is limited by severe cardiotoxicity. Free radicals, i.e., hydroxyl and superoxide radicals play a crucial role in this toxicity. In this study the involvement of the major metabolite of monoHER, 4'-O-methylmonoHER (methylmonoHER) in the protective effect of monoHER is studied. MethylmonoHER displayed antioxidant activity i.e., TEAC, hydroxyl and superoxide radical scavenging activity; nevertheless monoHER appeared to be superior compared to methylmonoHER. As a result of scavenging, flavonoids are oxidized and display reactivity towards thiols. Oxidized methylmonoHER, is far less thiol reactive towards creatine kinase than monoHER, which indicates that methylmonoHER is less toxic towards thiol containing enzymes. The thiol-reactivity of oxidized methylmonoHER was also negligible towards KEAP1 compared to monoHER. These results indicate that methylmonoHER hardly protects against radical damage via scavenging or via activating the NRF2 defense system. Also in HUVECs, methylmonoHER provided far less protection against oxidative stress (EC50>100µM) than monoHER which was a very potent protector (EC50=80nM). The results indicate that the contribution of methylmonoHER to the protection against doxorubicin-induced cardiotoxicity by monoHER is relatively low.