Molecular Detection of Toxoplasma gondii, Neospora caninum and Encephalitozoon spp. in Vespertilionid Bats from Central Europe.

Bats may carry various viruses and bacteria which can be harmful to humans, but little is known about their role as a parasitic source with zoonotic potential. The aim of this study was to test wild bats for the presence of selected parasites: Toxoplasma gondii, Neospora caninum and microsporidia Encephalitozoon spp. In total, brain and small intestine tissues of 100 bats (52 Myotis myotis, 43 Nyctalus noctula and 5 Vespertilio murinus) were used for the DNA isolation and PCR detection of the abovementioned agents. Toxoplasma gondii DNA was detected by real-time PCR in 1% of bats (in one male of M. myotis), while all bats were negative for N. caninum DNA. Encephalitozoon spp. DNA was detected by nested PCR in 25% of bats, including three species (twenty-two M. myotis, two N. noctula and one V. murinus). Positive samples were sequenced and showed homology with the genotypes Encephalitozoon cuniculi II and Encephalitozoon hellem 2C. This is the first study on wild vespertilionid bats from Central Europe and worldwide, with a relatively high positivity of Encephalitozoon spp. detected in bats.

Early recruitment of PARP-dependent m8A RNA methylation at DNA lesions is subsequently accompanied by active DNA demethylation.

RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

G-Quadruplex Structures Colocalize with Transcription Factories and Nuclear Speckles Surrounded by Acetylated and Dimethylated Histones H3.

G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.

Seroprevalence of Theileria equi, Babesia caballi, and Trichinella spp. infections in horses and donkeys from Nigeria, West Africa.

The protozoan parasites Theileria equi and Babesia caballi, transmitted by ticks, cause equine piroplasmosis, the most prevalent tick-borne disease in equids. Trichinellosis is a worldwide food-borne zoonosis caused by helminth Trichinella spp. that can lead to serious disease in humans, with fatal outcome. Although the infection is rare in horses, it deserves attention due to the increasing use of horse meat as a source of protein for humans. Horse trichinellosis is caused by several Trichinella species, most commonly by T. spiralis. The aim of the study was to determine the prevalence of antibodies to T. equi, B. caballi and Trichinella spp. in equids from three states of Northern Nigeria. Serum samples were collected from 139 clinically healthy animals, comprising 115 horses and 24 donkeys. Antibodies to T. equi and B. caballi were detected in serum by competitive-inhibition enzyme-linked immunosorbent assay (cELISA) and antibodies to Trichinella spp. by ELISA. Antibodies to T. equi were detected in 34% of equids (41% horses and 0% donkeys), antibodies to B. caballi in 9% of equids (8% horses and 13% donkeys), and antibodies to Trichinella spp. in 4% of equids (4% horses and 0% donkeys). There was co-infection of T. equi and B. caballi in 1% of horses and co-infection of T. equi and Trichinella spp. in 2.6% of horses. This is the first report on seroprevalence of Trichinella spp. in equids from Northern Nigeria.

A device for investigation of natural cell mobility and deformability.

A microfluidic device made of polydimethylsiloxane was developed for continuous evaluation of natural migration mobility of many eukaryotic cells in relaxed and deformed state. The device was fabricated by standard photolithography and soft lithography techniques using the SU-8 3010 negative photoresist on a glass wafer as the master mold. The simple flow-free device exploits the chemotactic movement of cells through a set of mechanical barriers in the direction of concentration gradients of attractants. The barriers are formed by arrays of circular cross-section pillars with decreasing spacing 7, 5, and 3 µm. To pass through the obstacles, the cells are deformed and change their cytoskeletal architecture. The instantaneous migration velocities of cells are monitored in a time-lapse setup of the scanning confocal microscope. Thus, the cellular deformability and migratory activity can easily be evaluated. The functionality of the device was tested with model HeLa cells stably transfected with fluorescent Premo FUCCI Cell Cycle Sensor. The designed device has the potential to be implemented for testing the tendency of patients' tumors to metastasis.

UVA irradiation strengthened an interaction between UBF1/2 proteins and H4K20 di-/tri-methylation.

Repair of ribosomal DNA (rDNA) is a very important nuclear process due to the most active transcription of ribosomal genes. Proper repair of rDNA is required for physiological biogenesis of ribosomes. Here, we analyzed the epigenetics of the DNA damage response in a nucleolar compartment, thus in the ribosomal genes studied in nonirradiated and UVA-irradiated mouse embryonic fibroblasts (MEFs). We found that the promoter of ribosomal genes is not abundant on H4K20me2, but it is densely occupied by H4K20me3. Ribosomal genes, regulated via UBF1/2 proteins, were characterized by an interaction between UBF1/2 and H4K20me2/me3. This interaction was strengthened by UVA irradiation that additionally causes a focal accumulation of H4K20me3 in the nucleolus. No interaction has been found between UBF1/2 and H3K9me3. Interestingly, UVA irradiation decreases the levels of H3K9me3 and H4K20me3 at 28S rDNA. Altogether, the UVA light affects the epigenetic status of ribosomal genes at 28S rDNA and strengthens an interaction between UBF1/2 proteins and H4K20me2/me3.

The Distinct Function and Localization of METTL3/METTL14 and METTL16 Enzymes in Cardiomyocytes.

It has become evident that epitranscriptome events, mediated by specific enzymes, regulate gene expression and, subsequently, cell differentiation processes. We show that methyltransferase-like proteins METTL3/METTL14 and N6-adenosine methylation (m6A) in RNAs are homogeneously distributed in embryonic hearts, and histone deacetylase (HDAC) inhibitors valproic acid and Trichostatin A (TSA) up-regulate METTL3/METTL14 proteins. The levels of METTL3 in mouse adult hearts, isolated from male and female animals, were lower in the aorta and pulmonary trunks when compared with atria, but METT14 was up-regulated in the aorta and pulmonary trunk, in comparison with ventriculi. Aging caused METTL3 down-regulation in aorta and atria in male animals. Western blot analysis in differentiated mouse embryonic stem cells (mESCs), containing 10-30 percent of cardiomyocytes, showed METTL3/METTL14 down-regulation, while the differentiation-induced increased level of METTL16 was observed in both wild type (wt) and HDAC1 depleted (dn) cells. In parallel, experimental differentiation in especially HDAC1 wild type cells was accompanied by depletion of m6A in RNA. Immunofluorescence analysis of individual cells revealed the highest density of METTL3/METTL14 in α-actinin positive cardiomyocytes when compared with the other cells in the culture undergoing differentiation. In both wt and HDAC1 dn cells, the amount of METTL16 was also up-regulated in cardiomyocytes when compared to co-cultivated cells. Together, we showed that distinct anatomical regions of the mouse adult hearts are characterized by different levels of METTL3 and METTL14 proteins, which are changed during aging. Experimental cell differentiation was also accompanied by changes in METTL-like proteins and m6A in RNA; in particular, levels and distribution patterns of METTL3/METTL14 proteins were different from the same parameters studied in the case of the METTL16 protein.

RETROSPECTIVE STUDY OF CAUSES OF ANIMAL MORTALITY IN LJUBLJANA ZOO 2005-2015.

Zoos harbor large collections of diverse species, aiding in both conservation and education, as well as research in multiple scientific fields. However, the most common causes of death in zoo animals around the world remain unclear because few extensive reports or reviews are published on this topic. This information could greatly improve preventive veterinary medicine in zoologic gardens. This study provides a retrospective overview of the causes of death of animals from the Ljubljana Zoo in the years 2005-2015. During this period, a total of 353 animals were submitted for necropsy, of which 244 were mammals, 85 were birds, and 25 were reptiles. The causes of deaths were divided into infectious diseases (38%), dysfunctions of individual organs (20%), traumas (13%), parasitosis (7%), reproductive disorders (6%), metabolic disorders (3%), neoplastic disease (4%), and intoxications (4%). In some cases, the cause of death was unable to be determined (7%), most commonly because of autolysis of the body. The results of this retrospective study bring a general overview of the epizootiologic situation in the Ljubljana Zoo over an 11-yr period and valuable information to other zoos to optimize preventative plans and diagnostics.

Toxoplasma gondii, Neospora caninum and Encephalitozoon cuniculi in Animals from Captivity (Zoo and Circus Animals).

Problems with parasitic infections are common in zoological gardens and circuses. In some animals it can lead to several disorders such as systemic disease, reproductive disorders (abortions and neonatal mortality), and even to death if severe illness is untreated. Thus, the aim of this study was to evaluate the prevalence of three common parasites in 74 animals from three zoos, and four circuses in Southern Italy. Antibodies to Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi were detected in 51%, 12%, and 20% of animals, respectively. Co-infections of T. gondii and N. caninum were reported in seven animals (9%) and co-infection of T. gondii and E. cuniculi in one animal. T. gondii, N. caninum and E. cuniculi seroprevalence differed in type of diet (P ≤ 0.0001; P ≤ 0.037 and P ≤ 0.004, respectively). T. gondii and E. cuniculi seroprevalence also differed in animal families (P ≤ 0.0001) and according to type of housing (P ≤ 0.003), respectively. Statistical differences were not found in other characteristics (gender, age, country of birth, origin, and contact with cats or dogs). This is the first serological study focusing on protozoan and microsporidian parasites in zoo and circus animals from Southern Italy and the first detection of antibodies to E. cuniculi in camels in Europe.

HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia-like animals.

Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1-deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro-differentiation was almost suppressed. Neuro-differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia-like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia-like brains that were treated with the cannabinoid receptor-1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co-regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro-differentiation as well as the pathophysiology of a schizophrenia-like phenotype.

Depletion of A-type lamins and Lap2α reduces 53BP1 accumulation at UV-induced DNA lesions and Lap2α protein is responsible for compactness of irradiated chromatin.

We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light. We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2α deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin. However, a well-known marker of double strand breaks, γH2AX, was highly abundant in the lesion-surrounding genome of Lap2α deficient cells. Described changes, induced by irradiation in Lap2α dn cells, were not accompanied by cell cycle changes. In Lap2α dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein. Two of them showed identical exponential decay times (τ1 and τ3), but the decay rate of τ2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2α dn fibroblasts. Moreover, γ-irradiation weakened an interaction between A-type lamins and Lap2α. Together, our results demonstrate how depletion of Lap2α affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2α deficient cells exposed to radiation.

Nuclear apoptotic volume decrease in individual cells: Confocal microscopy imaging and kinetic modeling.

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.

Tear production, intraocular pressure and conjunctival bacterial flora in selected captive wild ruminants.

OBJECTIVE: Evaluation of tear production (Schirmer's tear test, STT) and measurement of intraocular pressure (IOP) were performed in a population of captive wild ungulates in a Slovenian ZOO during routine annual health check. ANIMALS STUDIED: In total, 10 fallow deer (Dama dama), 25 mouflons (Ovis aries musimon), 20 alpine ibexes (Capra ibex), and three alpine chamois (Rupicapra rupicapra) were included in the study. METHODS: Tear production was performed by Schirmer's tear test, IOP was measured with an applanation tonometer, and ophthalmological examination was conducted with slit-lamp biomicroscopy and indirect ophthalmoscopy. Conjunctival swabs were taken and submitted for aerobic bacteriology and for detection of Chlamydia spp. and Mycoplasma spp. tested by PCR. RESULTS: Average tear production (in mm/min) was 17.8 ± 3.16 for fallow deer, 17.9 ± 3.87 for mouflons, and 11.7 ± 3.87 for ibexes. Mean intraocular pressure (IOP, in mm Hg) was 14.1 ± 2.48 for fallow deer, 14.9 ± 2.20 for mouflons, and 13.1 ± 2.43 for ibexes. For chamois, average tear production and IOP were 14.5 ± 3.0 and 10.2 ± 2.5, respectively; this is the first record of STT I and IOP in chamois. Bacteriological swabs were positive for bacteria in 100% of the fallow deer, 56% of mouflons, 35% of ibexes, and 100% of chamois. Gram-positive bacteria were predominant. Moraxella spp., Chlamydia spp., and Mycoplasma spp. were not detected. CONCLUSION: The reported values were obtained in animals under manual restraint only to be applicative in similar conditions.

Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells.

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.

Mutations in the TP53 gene affected recruitment of 53BP1 protein to DNA lesions, but level of 53BP1 was stable after γ-irradiation that depleted MDC1 protein in specific TP53 mutants.

53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.

Seroprevalence of Neospora caninum and Toxoplasma gondii in exotic ruminants and camelids in the Czech Republic.

Neospora caninum and Toxoplasma gondii are the protozoan parasites with definitive hosts from order Carnivora. Due to vertical transmission, both parasites can cause abortions and neonatal mortality that lead to significant productive and economic losses in the domestic ruminants. The aim of this study was to describe N. caninum and T. gondii seroprevalence in the group of frequently farmed captive exotic ruminants (n = 184) including Bovidae (barbary sheep, bezoar goat, common eland, American bison, water buffalo, and yak) and Camelidae (bactrian camel, guanaco, llama, and alpaca). Antibodies were tested by indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Higher prevalence of T. gondii antibodies (31% in IFAT and 52% in ELISA) was detected compared to N. caninum (24% in IFAT and 17% in cELISA). Mixed infection was found in 18 (10%) and 22 (12%) animals by IFAT and ELISA, respectively. Higher seroprevalence of both N. caninum and T. gondii was found in Camelidae compared to Bovidae. To author knowledge, this is the first detection of T. gondii and N. caninum antibodies in common elands and bezoar goats.

An eight-year survey of the intestinal parasites of carnivores, hoofed mammals, primates, ratites and reptiles in the Ljubljana zoo in Slovenia.

Problems with parasitic infections and their interspecies transmissions are common in zoological gardens and could pose serious health damage to captive animals. This study presents results of eight-year monitoring of intestinal parasites in animals from Zoo Ljubljana, Slovenia. A total of 741 faecal samples from 40 animal species were collected two to four times per year and examined microscopically. Intestinal parasites were detected in 45% of samples, with detection of helminths (Cestoda, Nematoda - Ascaridida, Enoplida, Strongylida, Oxyurida, Rhabditida and Trichurida) and protists (Apicomplexa and Ciliophora) in 25% and 13% of samples, respectively; mixed infection was found in 7% of samples. The mostly infected were ungulates (61%), followed by reptiles (44%), ratites (29%), primates (22%) and carnivores (7%). During the observation period, the number of infected animal species increased from 8 to 25. This is the first long-term monitoring study of intestinal parasites in zoo animals from Slovenia. Routine monitoring of parasitic infection and regular deworming and hygienic measures are necessary to prevent gastrointestinal infections in captive animals.

Localized Movement and Levels of 53BP1 Protein Are Changed by γ-irradiation in PML Deficient Cells.

We studied epigenetics, distribution pattern, kinetics, and diffusion of proteins recruited to spontaneous and γ-radiation-induced DNA lesions. We showed that PML deficiency leads to an increased number of DNA lesions, which was accompanied by changes in histone signature. In PML wt cells, we observed two mobile fractions of 53BP1 protein with distinct diffusion in spontaneous lesions. These protein fractions were not detected in PML-deficient cells, characterized by slow-diffusion of 53BP1. Single particle tracking analysis revealed limited local motion of 53BP1 foci in PML double null cells and local motion 53BP1 foci was even more reduced after γ-irradiation. However, radiation did not change co-localization between 53BP1 nuclear bodies and interchromatin granule-associated zones (IGAZs), nuclear speckles, or chromocenters. This newly observed interaction pattern imply that 53BP1 protein could be a part of not only DNA repair, but also process mediated via components accumulated in IGAZs, nuclear speckles, or paraspeckles. Together, PML deficiency affected local motion of 53BP1 nuclear bodies and changed composition and a number of irradiation-induced foci. J. Cell. Biochem. 117: 2583-2596, 2016. © 2016 Wiley Periodicals, Inc.

The level and distribution pattern of HP1ß in the embryonic brain correspond to those of H3K9me1/me2 but not of H3K9me3.

We studied the histone signature of embryonic and adult brains to strengthen existing evidence of the importance of the histone code in mouse brain development. We analyzed the levels and distribution patterns of H3K9me1, H3K9me2, H3K9me3, and HP1ß in both embryonic and adult brains. Western blotting showed that during mouse brain development, the levels of H3K9me1, H3K9me2, and HP1ß exhibited almost identical trends, with the highest protein levels occurring at E15 stage. These trends differed from the relatively stable level of H3K9me3 at developmental stages E8, E13, E15, and E18. Compared with embryonic brains, adult brains were characterized by very low levels of H3K9me1/me2/me3 and HP1ß. Manipulation of the embryonic epigenome through histone deacetylase inhibitor treatment did not affect the distribution patterns of the studied histone markers in embryonic ventricular ependyma. Similarly, Hdac3 depletion in adult animals had no effect on histone methylation in the adult hippocampus. Our results indicate that the distribution of HP1ß in the embryonic mouse brain is related to that of H3K9me1/me2 but not to that of H3K9me3. The unique status of H3K9me3 in the brain was confirmed by its pronounced accumulation in the granular layer of the adult olfactory bulb. Moreover, among the studied proteins, H3K9me3 was the only posttranslational histone modification that was highly abundant at clusters of centromeric heterochromatin, called chromocenters. When we focused on the hippocampus, we found this region to be rich in H3K9me1 and H3K9me3, whereas H3K9me2 and HP1ß were present at a very low level or even absent in the hippocampal blade. Taken together, these results revealed differences in the epigenome of the embryonic and adult mouse brain and showed that the adult hippocampus, the granular layer of the adult olfactory bulb, and the ventricular ependyma of the embryonic brain are colonized by specific epigenetic marks.