Current situation of lead (Pb) exposure in raptors and waterfowl in Japan and difference in sensitivity to in vitro lead exposure among avian species.

Although lead (Pb) poisoning in wild birds has been considered a serious problem in Japan for over 30 years, there is little information about Pb exposure and its sources throughout Japan except for Hokkaido. Furthermore, to identify and effectively prioritize the conservation needs of highly vulnerable species, differences in sensitivity to Pb exposure among avian species need to be determined. Therefore, we investigated the current situation of Pb exposure in raptors (13 species, N = 82), waterfowl (eight species, N = 44) and crows (one species, N = 6) using concentration and isotope analysis. We employed blood or tissue samples collected in various Japanese facilities mainly in 2022 or 2023. We also carried out a comparative study of blood δ-ALAD sensitivity to in vitro Pb exposure using blood of nine avian species. Pb concentrations in the blood or tissues displayed increased levels (>0.1 µg/g blood) in two raptors (2.4%), ten waterfowl (23%) and one crow (17%). Among them, poisoning levels (>0.6 µg/g blood) were found in one black kite and one common teal. The sources of Pb isotope ratios in ten blood samples with high Pb levels were determined as deriving from shot pellets (N = 9) or rifle bullets (N = 1). In the δ-ALAD study, red-crowned crane showed the highest sensitivity among the nine tested avian species and was followed in order by five Accipitriformes species (including white-tailed and Steller's sea eagle), Blakiston's fish owl, Muscovy duck and chicken, suggesting a genetically driven variance in susceptibility. Further studies on contamination conditions and exposure sources are urgently needed to inform strict regulations on the usage of Pb ammunition. Furthermore, detailed examinations of δ-ALAD sensitivity, interspecific differences, and other factors involved in the variability in sensitivity to Pb are required to identify and prioritize highly sensitive species.

Evaluation of anticoagulant rodenticide sensitivity by examining in vivo and in vitro responses in avian species, focusing on raptors.

Anticoagulant rodenticides (ARs) are used to control pest rodent species but can result in secondary poisoning of non-target animals, especially raptors. In the present study, differences in AR sensitivity among avian species were evaluated by comparing in vivo warfarin pharmacokinetics and effects, measuring cytochrome P450s (CYPs) expression involved in AR metabolism, and conducting in vitro inhibition assays of the AR target enzyme Vitamin K 2,3-epoxide reductase (VKOR). Oral administration of warfarin at 4 mg/kg body weight did not prolong prothrombin time in chickens (Gallus gallus), rock pigeons (Columba livia), or Eastern buzzards (Buteo japonicus). Rock pigeons and buzzards exhibited shorter plasma half-life of warfarin compared to chickens. For the metabolite analysis, 4'-hydroxywarfarin was predominantly detected in all birds, while 10-hydroxywarfarin was only found in pigeons and raptors, indicating interspecific differences in AR metabolism among birds likely due to differential expression of CYP enzymes involved in the metabolism of ARs and variation of VKOR activities among these avian species. The present findings, and results of our earlier investigations, demonstrate pronounced differences in AR sensitivity and pharmacokinetics among bird species, and in particular raptors. While ecological risk assessment and mitigation efforts for ARs have been extensive, AR exposure and adverse effects in predatory and scavenging wildlife continues. Toxicokinetic and toxicodynamic data will assist in such risk assessments and mitigation efforts.

Surveys of eleven species of wild and zoo birds and feeding experiments in white-tailed eagles reveal differences in the composition of the avian gut microbiome based on dietary habits between and within species.

The composition of the gut microbiome varies due to dietary habits. We investigated influences of diet on the composition of the gut microbiome using the feces of 11 avian species, which consumed grain-, fish- and meat-based diets. We analyzed gut microbiome diversity and composition by next-generation sequencing (NGS) of 16S ribosomal RNA. The grain-diet group had higher gut microbiome diversity than the meat- and fish-diet group. The ratio of Bacteroidetes and Firmicutes phyla was higher in the grain-diet group than in the meat- and fish-diet groups. The grain-diet group had a higher ratio of Veillonellaceae than the meat-diet group and a higher ratio of Eubacteriaceae than the fish-diet habit group. To clarify the influence of diet within the same species, white-tailed eagles (Haliaeetus albicilla, n=6) were divided into two groups, and given only deer meat or fish for approximately one month. The composition of the gut microbiome of individuals in both groups were analyzed by NGS. There were indications of fluctuation in the levels of some bacteria (Lactobacillus, Coriobacteriales, etc.) in each diet group. Moreover, one individual for each group which switched each diet in last week changed to each feature of composition of bacterial flora. The above results show that the composition of the gut microbiome differ depending on diet, even within the same species.

A novel reagent for the screening of haptoglobin-deficient blood donors.

BACKGROUND AND OBJECTIVES: In Japan, the prevalence of haptoglobin deficiency is approximately 1 in 4000. Haptoglobin-deficient individuals may produce anti-haptoglobin from allo-immunization, leading to serious transfusion reactions. Therefore, implementation of a consistent supply of haptoglobin-deficient fresh frozen plasma is crucial. We developed a novel reagent to facilitate large-scale identification of haptoglobin-deficient individuals as potential donors of plasma products. MATERIALS AND METHODS: We established mouse monoclonal anti-haptoglobin-producing cell lines (three clones) using the hybridoma method by immunizing mice with the haptoglobin protein. Purified antibodies were conjugated with carboxylate-modified polystyrene latex beads and used for haptoglobin measurements by the latex agglutination method using an automatic analyser (LABOSPECT008). Samples with low protein concentrations were re-examined by enzyme-linked immunosorbent assay to confirm the results. Additionally, the haptoglobin gene was amplified by polymerase chain reaction to confirm the haptoglobin deletion allele (Hpdel ). RESULTS: From February to October 2022, 7476 blood donor samples were screened. Two haptoglobin-deficient and 21 low-haptoglobin-expressing individuals were identified. Two haptoglobin-deficient donors were found homozygous for Hpdel , and 19 (90%) of the 21 low-haptoglobin-expressing individuals were heterozygous for Hpdel , which includes the first reported case of heterozygous Hpdel /HpJohnson . CONCLUSION: We developed a new reagent for the detection of haptoglobin deficiency, which is automatable and inexpensive and appears useful for large-scale screening of blood donors.

Determinants of Attitude Toward End-of-Life Care Among Junior Physicians: Findings from a Nationwide Survey in Japan.

Background: Physicians' attitudes can be critical in quality end-of-life care. However, the determinants of the attitudes and whether the attitudes can be modified remain unclear. Objectives: To investigate factors correlated with physicians' positive attitudes toward end-of-life care and whether these attitudes are modifiable through acquired factors (e.g., education or mentorship). Design: A nationwide survey was conducted in 300 institutions and selected randomly from 1037 clinical training hospitals in Japan. Participants: From each selected institution, two resident physicians of postgraduate year (PGY) 1 or 2 and two clinical fellows from PGY 3-5 were requested to answer the survey. Measurements: The primary outcome was the Frommelt Attitudes Toward the Care of the Dying (FATCOD) scale score. Factors (e.g., the respondents' age, sex, number of years of clinical experience, training environment, religion, and beliefs around death) were examined for correlation with FATCOD score. Results: In all, 198 physicians and 134 clinical fellows responded to the survey (response rate: 33.0% and 22.3%, respectively). Factors with the strongest correlation with FATCOD scores were mostly unmodifiable factors (e.g., being female and one's beliefs around death). Modifiable factors were also identified-number of patient deaths experienced, level of interest in palliative care, availability of support from senior mentors, and frequency of consultation with nonphysician medical staff. Conclusion: Physicians' attitudes toward end-of-life care correlate more strongly with nonmodifiable factors, but attitudes can be meaningfully improved via mentoring by senior physicians. Future studies are warranted to determine the effective interventions to foster positive attitudes among physicians involved in end-of-life care.

Induced pluripotent stem cells of endangered avian species.

The number of endangered avian-related species increase in Japan recently. The application of new technologies, such as induced pluripotent stem cells (iPSCs), may contribute to the recovery of the decreasing numbers of endangered animals and conservation of genetic resources. We established novel iPSCs from three endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston's fish owl) with seven reprogramming factors (M3O, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2). The iPSCs are pluripotency markers and express pluripotency-related genes and differentiated into three germ layers in vivo and in vitro. These three endangered avian iPSCs displayed different cellular characteristics even though the same reprogramming factors use. Japanese ptarmigan-derived iPSCs have different biological characteristics from those observed in other avian-derived iPSCs. Japanese ptarmigan iPSCs contributed to chimeras part in chicken embryos. To the best of our knowledge, our findings provide the first evidence of the potential value of iPSCs as a resource for endangered avian species conservation.

Experimental and natural infections of white-tailed sea eagles (Haliaeetus albicilla) with high pathogenicity avian influenza virus of H5 subtype.

White-tailed sea eagle (Haliaeetus albicilla), a regionally rare species of raptor, is threatened in several countries. To assess the risk of H5 high pathogenicity avian influenza (HPAI) viral infection in rare bird species, we performed experimental infections with a GS/GD96-lineage H5N6 HPAI virus of clade 2.3.4.4e in white-tailed sea eagles. Additionally, during the winter of 2020-2021 in Japan, we accidentally encountered a white-tailed sea eagle that had a fatal outcome due to natural infection with a GS/GD96-lineage H5N8 HPAI virus of clade 2.3.4.4b, allowing us to compare experimental and natural infections in the same rare raptor species. Our experiments demonstrated the susceptibility of white-tailed sea eagles to the GS/GD96-lineage H5 HPAI virus with efficient replication in systemic organs. The potential for the viruses to spread within the white-tailed sea eagle population through indirect transmission was also confirmed. Comprehensive comparisons of both viral distribution and histopathological observations between experimentally and naturally infected white-tailed sea eagles imply that viral replication in the brain is responsible for the disease severity and mortality in this species. These findings provide novel insights into the risk assessment of H5 HPAI viral infection in white-tailed sea eagles, proper diagnostic procedures, potential risks to artificially fed eagle populations and persons handling superficially healthy eagles, potential impact of intragastric infection on eagle outcomes, and possibility of severity of the disease being attributed to viral replication in the brain.

Behavior and toxic effects of Pb in a waterfowl model with oral exposure to Pb shots: Investigating Pb exposure in wild birds.

Among wild birds, lead (Pb) exposure caused by ingestion of ammunition is a worldwide problem. We aimed to reveal the behavior and toxic effect of Pb caused by ingesting Pb shots in waterfowl. Four male, eight-week old Muscovy ducks (Cairina moschata) were given three Pb shots (approximately 240 mg in total) orally and then fed for 29 days after exposure, simulating a low-dose Pb exposure in wild waterfowl. During the breeding period, blood samples were collected 10 times, and fecal samples every day. Additionally, 22 fresh tissue and 6 bone samples were obtained from each duck through the dissection. Although there were no gross abnormalities, the maximum blood Pb concentration of each duck ranged from 0.6 to 3.7 mg/L, reaching a threshold concentration indicative of clinical symptoms (>0.5 mg/L). δ-aminolevulinic acid dehydratase declined one day after exposure and remained low throughout the feeding period. Hematocrit also tended to decrease, indicating signs of anemia. The highest Pb accumulation was observed in the bones, followed by the kidneys, intestinal tracts, and liver. High Pb accumulation in the bones, which are known to have a long Pb half-life, suggested that Pb would remain in the body and possibly affect bird health beyond 28 days after exposure. Gene expression analysis showed a significant increase in the expression of the toll-like receptor-3 gene, which is involved in virus discrimination in the liver, suggesting a disruption of the immune system. Microbiota analyses showed a correlation between the blood Pb concentration and the abundances of Lachnospiraceae and Ruminococcaceae, suggesting that Pb affects lipid metabolism. These results provide fundamental data on Pb exposure in wild birds and a new perspective on the damage such exposure causes.

Analysis of lead distribution in avian organs by LA-ICP-MS: Study of experimentally lead-exposed ducks and kites.

Lead poisoning of wild birds by ingestion of lead ammunition occurs worldwide. Histopathological changes in organs of lead-intoxicated birds are widely known, and lead concentration of each organ is measurable using mass spectrometry. However, detailed lead localization at the suborgan level has remained elusive in lead-exposed birds. Here we investigated the detailed lead localization in organs of experimentally lead-exposed ducks and kites by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). In both the ducks and kites, lead accumulated diffusely in the liver, renal cortex, and brain. Lead accumulation was restricted to the red pulp in the spleen. With regard to species differences in lead distribution patterns, it is noteworthy that intensive lead accumulation was observed in the arterial walls only in the kites. In addition, the distribution of copper in the brain was altered in the lead-exposed ducks. Thus, the present study shows suborgan lead distribution in lead-exposed birds and its differences between avian species for the first time. These findings will provide fundamental information to understand the cellular processes of lead poisoning and the mechanisms of species differences in susceptibility to lead exposure.

Molecular characterization of Trichomonas gypaetinii isolated from the upper alimentary tract of Steller's sea eagles (Haliaeetus pelagicus) and white-tailed sea eagles (Haliaeetus albicilla) in Hokkaido, Japan.

Recent phylogenetic and morphologic studies of Trichomonas spp. suggests that there are more than 3 species that infect the upper alimentary tract of wild birds, which include T. gallinae, T. stableri, and T. gypaetinii. In this study, investigations were conducted on the prevalence of trichomonads in the upper alimentary tract of 12 Steller's sea eagles (Haliaeetus pelagicus) and 18 white-tailed sea eagles (H. albicilla). All birds were rescued from the wild and kept at a rehabilitation facility in Hokkaido, Japan, for variable durations and did not show any symptoms of trichomonosis. The ITS1-5.8SrRNA-ITS2 (ITS) genomic region of Trichomonas spp. was detected from 29 samples by PCR, and flagellates were confirmed from 4 samples by culture. Morphologic observations and measurement recordings were conducted under a light microscope on trophozoites obtained from the cultured isolates. Genomic sequences of the ITS, 18S ribosomal RNA (18S rRNA), Fe-hydrogenase, and RNA polymerase II largest subunit (Rpb1) regions were determined by direct sequencing, and phylogenetic analyses were conducted with previously published sequences of Trichomonas spp. All isolates were concluded as T. gypaetinii based on morphologic and molecular characterizations of the ITS and 18S rRNA genes. This is the first study to isolate T. gypaetinii from Haliaeetus eagles and further provide novel sequences of the Fe-hydrogenase and Rpb1 genes of T. gypaetinii. Both genomic regions also confirmed that T. gypaetinii belong to independent clusters from other Trichomonas spp.

Re-Invasion of H5N8 High Pathogenicity Avian Influenza Virus Clade 2.3.4.4b in Hokkaido, Japan, 2020.

Global dispersion of high pathogenicity avian influenza (HPAI), especially that caused by H5 clade 2.3.4.4, has threatened poultry industries and, potentially, human health. An HPAI virus, A/northern pintail/Hokkaido/M13/2020 (H5N8) (NP/Hok/20) belonging to clade 2.3.4.4b, was isolated from a fecal sample collected at a lake in Hokkaido, Japan where migratory birds rested, October 2020. In the phylogenetic trees of all eight gene segments, NP/Hok/20 fell into in the cluster of European isolates in 2020, but was distinct from the isolates in eastern Asia and Europe during the winter season of 2017-2018. The antigenic cartography indicates that the antigenicity of NP/Hok/20 was almost the same as that of previous isolates of H5 clade 2.3.4.4b, whereas the antigenic distances from NP/Hok/20 to the representative strains in clade 2.3.4.4e and to a strain in 2.3.4 were apparently distant. These data imply that HPAI virus clade 2.3.4.4b should have been delivered by bird migration despite the intercontinental distance, although it was not defined whether NP/Hok/20 was transported from Europe via Siberia where migratory birds nest in the summer season. Given the probability of perpetuation of transmission in the northern territory, periodic updates of intensive surveys on avian influenza at the global level are essential to prepare for future outbreaks of the HPAI virus.

Current situation regarding lead exposure in birds in Japan (2015-2018); lead exposure is still occurring.

Birds of a number of species have died as a result of lead (Pb) poisoning, including many Steller's sea eagles (Haliaeetus pelagicus) and white-tailed sea eagles (Haliaeetus albicilla) in Hokkaido, the northernmost island of Japan. To address this issue, the use of any type of Pb ammunition for hunting of large animals was prohibited in Hokkaido in 2004. However, Pb poisoning is still being reported in this area, and there are few regulations regarding the use of Pb ammunition in other parts of Japan, where it has been reported that eagles and water birds have been exposed to Pb. This study was performed to accurately determine the current level of Pb exposure of birds found dead in the field or dead in the wild bird centers in Japan (June 2015-May 2018) and to identify the sources of Pb. Pb exposure was found to still be occurring in raptors and water birds in various parts of Japan. Twenty-six point five % and 5.9% of the recorded deaths of Steller's sea eagles and white-tailed sea eagles, respectively, were found to have been poisoned by Pb. In addition, Pb isotope ratio analysis showed that both Pb rifle bullets and Pb shot pellets cause Pb exposure in birds, and these endangered eagles are also exposed to Pb in Hokkaido due to the illegal use of Pb ammunition. Changing to Pb-free ammunition, such as copper (Cu) rifle bullets, steel shot pellets, or bismuth shot pellets, will be essential for the conservation of avian species in Japan.

Microminipig: A suitable animal model to estimate oral absorption of sustained-release formulation in humans.

We investigated the gastrointestinal absorption characteristics of oral sustained-release formulations in microminipigs, dogs, and monkeys in order to clarify the similarities in absorption properties between these animals and humans. Time profiles of oral absorption of nifedipine and valproic acid were calculated from the plasma concentration-time profiles of the drugs by a deconvolution method. The curves for both drugs in microminipigs were close to or slightly higher than those in humans, whereas those in monkeys were lower. Furthermore, the plasma concentration-time profiles of the drugs were subjected to non-compartmental analysis. The fractions of a dose absorbed into the portal vein (FaFg) in microminipigs ranged from 50 to 100% of the human values, whereas those in monkeys were less than half the human values. In addition, the other absorption-related parameters for the sustained-release formulation in microminipigs, as well as monkeys, were comparable to those in humans. In conclusion, the oral absorption properties of microminipigs and humans were similar regarding the sustained-release formulations. Therefore, microminipig is a suitable animal model to estimate the oral absorption of sustained-release formulations in humans.

Epidemiologic Survey of Avian Influenza Virus Infection in Shorebirds Captured in Hokkaido, Japan.

There is limited information about virus epidemiology of shorebirds (family Charadriidae and Scolopacidae) in the East Asia-Australasia flyway. We investigated the prevalence of avian influenza viruses (AIVs) in shorebirds in Hokkaido, Japan, the stopover site of the flyway, to understand the ecology of AIV translocation in the flyway from 2006 to 2010. In total, 1,698 shorebirds belonging to 26 species were captured and released into two different sites using mist nets. Tracheal and cloacal swabs were collected from each bird using cotton swabs. The RNA of influenza A viruses was detected using reverse transcription loop-mediated isothermal amplification. One AIV-positive sample was obtained from a Lesser Sand Plover (Charadrius mongolus) captured in September 2010 at Lake Komuke. Full lengths of hemagglutinin (HA), neuraminidase (NA), polymerase acidic protein, nucleoprotein, matrix protein 1, and nuclear export protein genes were successfully amplified from the AIV-positive sample. All sequences showed the highest identity with sequences obtained from virus strains from Anseriformes species. Shorebirds migrated to Japan 1 mo earlier than did Anseriformes species. Therefore, the Lesser Sand Plover could have been infected by the virus from Anseriformes species on the breeding grounds. The HA sequence showed the highest identity with the H10 sequence whereas the NA sequence exhibited the highest identity with the N7 sequence. Phylogenic analysis showed that the detected subtype H10N7 belongs to the Eurasia lineage and the related strain might have widely spread in Asia in 2009.

Avian interspecific differences in VKOR activity and inhibition: Insights from amino acid sequence and mRNA expression ratio of VKORC1 and VKORC1L1.

Worldwide use of anticoagulant rodenticides (ARs) for rodents control has frequently led to secondary poisoning of non-target animals, especially raptors. In order to suggest some factors that may help considering the mechanism of the incidents, this study focused on the avian vitamin K 2, 3-epoxide reductase (VKOR) that is the target protein of ARs. We addressed the interspecific differences in VKOR activity and inhibition related to amino acid sequence and mRNA expression of VKORC1 and VKORC1-like1 (VKORC1L1). Poultry have been considered to be more tolerant to ARs than mammals. However, VKOR activity of owls, hawks, falcon and surprisingly, canaries, was lower and inhibited by warfarin more easily than that of chickens and turkeys. The amino acid sequence of VKORC1 and VKORC1L1 implied that the value of Ki for VKOR activity to ARs could depend on the amino acid at position 140 in the TYX warfarin-binding motif in VKORC1, and other amino acid mutations in VKORC1L1. The mRNA expression ratio of VKORC1:VKORC1L1 differed between turkey (8:1) and chicken (2:3) liver. VKORC1L1 has been reported to be resistant to warfarin compared to VKORC1. Hence, both the Ki of specific VKORC1 and VKORC1L1, and the mRNA expression ratio would cause avian interspecific difference of the VKOR inhibition. Our study also suggested the high inhibition of VKOR activities in raptors and surprisingly that in canaries as well. These factors are the most likely to contribute to the high sensitivity to ARs found in raptors.

Poxvirus infection in a Steller's sea eagle (Haliaeetus pelagicus).

A severely emaciated adult Steller's sea eagle (Haliaeetus pelagicus) was found dead with electrocution-induced severe wing laceration, and with multiple cutaneous pock nodules at the periocular regions of both sides nearby the medial canthi and rhamphotheca. Histopathological examination of the nodules revealed hyperplasia of the epidermis with vacuolar degeneration and intracytoplasmic inclusion bodies (Bollinger bodies). The proventriculus was severely affected by nematodes and was ulcerated. Nucleotide sequencing of a PCR-amplified product of Avipoxvirus 4b core gene revealed 100% identity to the sequence of Avipoxvirus derived from other eagle species. This report describes the first detection of Avipoxvirus clade A from a Steller's sea eagle.

Characterization and imaging of lead distribution in bones of lead-exposed birds by ICP-MS and LA-ICP-MS.

Lead (Pb) poisoning in raptors and water birds is a serious problem in many countries. However, only a small fraction of Pb poisoning has been detected in birds. Bone specimens may be useful indices of Pb exposure because bones contain ∼90% of the total Pb body burden. The original purpose of this study was to comprehensively analyze Pb accumulation in various bone types using inductively coupled plasma-mass spectrometry (ICP-MS). Since our results showed that Pb accumulation differed greatly depending on bone type, a secondary objective was defined, aiming to investigate the fine Pb distribution and its relation to bone structure and bone marrow by using laser ablation (LA)-ICP-MS. Our findings suggested that bone samples (1) consisting of trabecular tissue and (2) those that contain bone marrow could accumulate high levels of Pb following acute exposure. The shorter turnover time of trabecular bone can cause a rapid accumulation of Pb, and bone marrow may have an important role for internal exposure of Pb to bone tissue. Pb is accumulated in bones via blood flow, and bone marrow receives blood from outside the bones. In conclusion, bone samples provide valuable information on Pb exposure and could be useful to investigate and understand mortalities related to suspected Pb poisoning.

Lead exposure in raptors from Japan and source identification using Pb stable isotope ratios.

Lead (Pb) poisoning is widespread among raptors and water birds. In Japan, fragments of Pb ammunition are still found in endangered eagles although more than 10 years have passed since legislation regarding use of Pb ammunition was introduced. This study was performed to investigate Pb exposure in raptors from various locations in Japan. We measured hepatic and renal Pb concentrations and hepatic Pb isotope ratios of Steller's sea eagles (Haliaeetus pelagicus), white-tailed sea eagles (Haliaeetus albicilla), golden eagles (Aquila chrysaetos), and 13 other species (total 177 individuals) that were found dead, as well as blood samples from three eagles found in a weakened state during 1993-2015 from Hokkaido (northern part), Honshu (the main island), and Shikoku (a southern island) of Japan. In the present study in Hokkaido, one quarter of the sea eagles showed a high Pb concentration, suggesting exposure to abnormally high Pb levels and Pb poisoning. Pb isotope ratios indicated that endangered Steller's sea eagle and white-tailed sea eagle were poisoned by Pb ammunition that was used illegally in Hokkaido. In other areas of Japan, both surveillance and regulations were less extensive than in Hokkaido, but Pb poisoning in raptors was also noted. Therefore, Pb poisoning is still a serious problem in raptors in various areas of Japan due to accidental ingestion of materials containing Pb, especially Pb ammunition.

Spatial Interplay between Polycomb and Trithorax Complexes Controls Transcriptional Activity in T Lymphocytes.

Trithorax group (TrxG) and Polycomb group (PcG) proteins are two mutually antagonistic chromatin modifying complexes, however, how they together mediate transcriptional counter-regulation remains unknown. Genome-wide analysis revealed that binding of Ezh2 and menin, central members of the PcG and TrxG complexes, respectively, were reciprocally correlated. Moreover, we identified a developmental change in the positioning of Ezh2 and menin in differentiated T lymphocytes compared to embryonic stem cells. Ezh2-binding upstream and menin-binding downstream of the transcription start site was frequently found at genes with higher transcriptional levels, and Ezh2-binding downstream and menin-binding upstream was found at genes with lower expression in T lymphocytes. Interestingly, of the Ezh2 and menin cooccupied genes, those exhibiting occupancy at the same position displayed greatly enhanced sensitivity to loss of Ezh2. Finally, we also found that different combinations of Ezh2 and menin occupancy were associated with expression of specific functional gene groups important for T cell development. Therefore, spatial cooperative gene regulation by the PcG and TrxG complexes may represent a novel mechanism regulating the transcriptional identity of differentiated cells.