Antizyme Inhibitor 2-Deficient Mice Exhibit Altered Brain Polyamine Levels and Reduced Locomotor Activity.

BACKGROUND: Alterations in the neural polyamine system are known to be associated with different brain pathological conditions. In addition, the regulation of enzymes involved in polyamine metabolism such as ornithine decarboxylase (ODC), antizymes (AZs), and antizyme inhibitors (AZINs) is critical during brain development. However, while most studies focus on ODC and AZs, less is known about AZIN expression and function in the brain. Thus, our aim was to analyze the expression pattern of AZIN2 during postnatal development, its brain distribution, and its possible implication in phenotypical alterations. METHODS: The expression pattern of Azin2 and other genes related to polyamine metabolism was analyzed by RT-qPCR. ß-D-galactosidase staining was used to determine the anatomical distribution of AZIN2 in a Azin2 knockout model containing the ßGeo marker. Brain polyamine content was determined by HPLC. The Rota-Rod and Pole functional tests were used to evaluate motor skills in Azin2-lacking mice. RESULTS: Our results showed that expression of genes codifying for AZs and AZINs showed a similar increasing pattern over time that coincided with a decrease in ODC activity and putrescine levels. The analysis of AZIN2 distribution demonstrated that it is strongly expressed in the cerebellum and distributed along the neuron body and dendrites. The ablation of Azin2 showed a decrease in putrescine levels and is related to reduced motor skills. CONCLUSIONS: Our study revealed that AZIN2 expression in the brain is particularly limited to the cerebellum. In addition, the ablation of Azin2 leads to a reduction in putrescine that relates to alterations in motor function, suggesting the role of AZIN2 in the functioning of dopaminergic neurons.

ATR expands embryonic stem cell fate potential in response to replication stress.

Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS.

Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress.

The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

Histone H2A C-terminus regulates chromatin dynamics, remodeling, and histone H1 binding.

The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A C-terminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformation.

Molecular and morphological changes in placenta and embryo development associated with the inhibition of polyamine synthesis during midpregnancy in mice.

Polyamines play an essential role in murine development, as demonstrated by both gene ablation in ornithine decarboxylase (ODC)-deficient embryos and pharmacological treatments of pregnant mice. However, the molecular and cellular mechanisms by which ODC inhibition affects embryonic development during critical periods of pregnancy are mostly unknown. Our present results demonstrate that the contragestational effect of alpha-difluoromethylornithine (DFMO), a suicide inhibitor of ODC, when given at d 7-9 of pregnancy, is associated with embryo growth arrest and marked alterations in the development of yolk sac and placenta. Blood island formation as well as the transcript levels of embryonary globins alpha-like x chain and beta-like y-chain was markedly decreased in the yolk sac. At the placental level, abnormal chorioallantoic attachment, absence of the spongiotrophoblast layer and a deficient development of the labyrinthine zone were evident. Real-time RT-PCR analysis showed that transcript levels of the steroidogenic genes steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase VI, and 17alpha-hydroxylase were markedly decreased by DFMO treatment in the developing placenta at d 9 and 10 of pregnancy. Plasma values of progesterone and androstenedione were also decreased by DFMO treatment. Transcriptomic analysis also detected changes in the expression of several genes involved in placentation and the differentiation of trophoblastic lineages. In conclusion, our results indicate that ODC inhibition at d 8 of pregnancy is related to alterations in yolk sac formation and trophoblast differentiation, affecting processes such as vasculogenesis and steroidogenesis.