Glaciers and ice sheets, like other biomes, occupy a significant area of the planet and harbour biological communities with distinct interactions and feedbacks with their physical and chemical environment. In the case of the glacial biome, the biological processes are dominated almost exclusively by microbial communities. Habitats on glaciers and ice sheets with enough liquid water to sustain microbial activity include snow, surface ice, cryoconite holes, englacial systems and the interface between ice and overridden rock/soil. There is a remarkable similarity between the different specific glacial habitats across glaciers and ice sheets worldwide, particularly regarding their main primary producers and ecosystem engineers. At the surface, cyanobacteria dominate the carbon production in aquatic/sediment systems such as cryoconite holes, while eukaryotic Zygnematales and Chlamydomonadales dominate ice surfaces and snow dynamics, respectively. Microbially driven chemolithotrophic processes associated with sulphur and iron cycle and C transformations in subglacial ecosystems provide the basis for chemical transformations at the rock interface under the ice that underpin an important mechanism for the delivery of nutrients to downstream ecosystems. In this review, we focus on the main ecosystem engineers of glaciers and ice sheets and how they interact with their chemical and physical environment. We then discuss the implications of this microbial activity on the icy microbiome to the biogeochemistry of downstream ecosystems. One of the major limiting factors for the development of microbial processes is the presence of liquid water. The overwhelming presence of snow and ice does not preclude glaciers and ice sheets of containing large amounts of surface and interstitial water for long periods during the melt season. The annual runoff from Greenland alone is estimated to be around 1015 litres,2 while the amount of water stored as ice and snow globally is around 1019 litres. Bacterial cell abundances in the wet areas of glaciers vary significantly between 107 cells L−1 in clean snow and ice3 to 1011 cells L−1 in surface ice associated with impurities.5 We know little about the rate or types of microbes delivered onto remote snow and ice fields through Aeolian processes8 and about microbial abundances in subglacial habitats as these are far more difficult to sample. For subglacial systems, recent measurements of bacterial numbers associated with sediments from glaciers in the Arctic and Antarctica and subglacial runoff range between 0.87 and 7.9 × 106 cells g−1 .Due to the background geothermal heat and the pressure of the ice that keeps liquid water above the pressure melting point, water can also form beneath glaciers and ice sheets Figs e and 2a.1 They are responsible for the accumulation of organic matter at the surface of glaciers, which in turn leads to positive feedbacks between melting and microbial activity. Organic matter produced at the surface of the snow and ice by those ecosystem engineers can also be transported through the englacial system down to subglacial environments, fuelling microbial processes under the ice. In subglacial environments, several chemolithotrophic processes have been described providing an additional pathway by which microbial heterotrophic activity is sustained. In this review, we give particular emphasis on the primary producers of different glacial habitats.Several recent reviews have described the microbial ecology of the different habitats of glaciers and ice sheets  that thrive in glacial snow and permanent snow fields , suggesting a cosmopolitan occurrence for these organisms. The first descriptions of snow algal taxonomy, ecology and physiology were based on classical microscopy observations by Kol35 and Ettl36 and later by Hoham et al.37 and Komárek and Nedbalová.38 The red snow  phenomenon has been known for a long time and has already been described by the Ancient Greek Aristotle.39 Yet, only in the 19th century the development of microscopy allowed scientists to link red snow to biological processes, namely snow algae blooms, and this work led to the naming of the taxon Chlamydomonas nivalis by Wille.40 Since then a large number of snow algal taxa have been described and wrongly classified as Chlamydomonas nivalis. Kol35was the first to state that this taxon has to be regarded as a collective name and that it is not the only species that causes the colouring. The same confusion applies for the taxon Chloromonas nivalis that cannot be regarded as one species either, as revealed by genetic analyses.23 Although important, this classification problem is still often neglected nowadays.Snow algae are a group of unicellular freshwater algae  chlorophyll-rich trophic stages of the latter red snow taxa42 or an independent phenomenon caused by the genera Microglena27 or Raphidonema.35 However, Raphidonema sempervirens and R. nivale (Trebouxiophyceae) are typical permafrost algae and not true snow algal taxa. Laboratory experiments43 also demonstrated that Raphidonema sempervirens produces primary , but not secondary  carotenoids, that are characteristic of the red snow cysts. Raphidonema sempervirens use the xanthophyll cycle to dissipate excessive energy as heat through non-photochemical quenching.44 Furthermore, Raphidonema cells are likely introduced onto glacial surfaces by wind rather than through in situ propagation.45 Thus, it remains unclear, whether this taxon is a critical or a minor player in the ecology of glaciers. Finally, less often described (and probably less abundant) is yellow snow caused by Hydrurus related Chrysophyceae.46Snow algae can form extensive blooms in summer after the onset of melting. In response to extensive light exposure, they develop pigmentation as an adaptation mechanism. Depending on the concentration and composition of the produced pigments, the result is a macroscopically visible colouration of the snow. This can vary from green (chlorophylls) to different shades of yellow (xantophylls), orange and red .47 concluded that ecosystem chemistry may only play a minor role in comparison to physical parameters, which includes field topography  that impacts on melting. Using molecular identification, Lutz et al.47 found similar snow algal community composition across the Arctic that is independent of location-specific geochemical factors, with an uncultured Chlamydomonadaceae taxon showing the highest relative abundance. On the other hand, Brown et al.48 showed that on the haplotype level, snow algal populations are locally heterogeneous and each population was derived from one clone. Whereas at the genus and most often species level the above mentioned taxa can be found in the Arctic, Antarctic and the European Alps, future work needs to investigate their similarities or differences on the sub-species level as well as their distribution patterns on different scales .The parameters controlling snow algal distribution are far from being well understood. Several studies37 and Remias,42 who suggested that Chlamydomonas nivalis cells remain dormant as cysts for most of the year. At the onset of melting in spring flagellated cells emerge and migrate to the snow surface where they start to bloom. However, this migration process has not yet been fully observed or documented in field settings and needs further investigation. During the melt season, cells undergo a variety of intracellular rearrangements and changes in morphology . These stages are likely linked to nutrient limitation, as under nitrogen-deficiency the metabolism is directed towards nitrogen-free metabolites such as lipids  and pigments .43 Astaxanthin, the main secondary carotenoid, is often esterified with fatty acids and stored in cytoplasmatic lipid droplets around the nucleus and chloroplast, which in turn also results in their shading from high light irradiation.49 Thus, protection against excessive radiation could also involve the accumulation of primary and secondary carotenoids (to shield the chloroplast), which enhances lipid production (less cytoplasmatic water content), resulting in reinforcement of the cell wall and cyst formation as one of the suit of adaptation mechanisms for organic carbon production in snow. Snow algae are actively fixing carbon in these ecosystems as demonstrated by net ecosystem production measurements.4 The potential for labile organic carbon production in snow is reflected in bacterial abundance and production rates, that were found to be higher in red snow containing algae compared to white snow with low snow algal numbers.50 This suggests that snow algae are an important source of organic matter. Similarly, Brown et al.51 found a co-occurrence of snow algae and fungi but no studies have been conducted to investigate the nature of their interaction.The main stages in snow algal life cycles have been described in Hoham et al.37 and these are of wide interest for biotechnological application.52 A large number of cultured snow algal strain is available from CCCryo (Culture Collection of Cryophilic Algae http://cccryo.fraunhofer.de/web/infos/welcome/), which serves as a valuable bioresource for laboratory experiments  decrease the albedo of snow and ice surfaces, which in turn may speed up melting processes.8 they impart glacial surfaces a brownish-greyish colouration that is often mistaken for ‘‘dirt’’. Although these blooms had already been mentioned in the 1870s by Adolf Erik Nordenskiöldii and Sven Berggren during expeditions to Greenland,58 ice algae have only recently been described in detail.61 Ice algal mass blooms are believed to show cosmopolitan occurrence in many permanently frozen alpine and polar settings63 and they are thought to be important primary producers on ice surfaces,61 yet much about their ecosystem function is still unknown. The “true” ice algal species, that are well adapted to temperatures around the freezing point and that would survive in warmer places only temporarily, are the filamentous Ancylonema nordenskiöldii Berggren 1871 and the two unicellular species, Mesotaenium berggrenii Lagerheim 1892 and Cylindrocystis brebissonii fo. Cryophila Kol 1942 -A and UV-B range and, therefore, may play an important adaptation role in shielding the chloroplast and avoiding photoinhibition. The same pigmentation has been found in Mesotaenium berggrenii cells thriving in less UV irradiated settings, suggesting that this compound may additionally act as an antimicrobial agent in addition to its photoprotective role.66 Primary carotenoids have been identified, whereas secondary carotenoids seem to be absent.60 Due to their dark brown pigmentation and their wide occurrence, glacial ice algae play an important role in reducing the albedo of bare ice fields during summer and thus in increasing glacial melting.63Ice algal taxa are restricted to a very short growth season when liquid water is available and the snow cover is gone. The rest of the year they have to cope with desiccation, darkness and temperature stress. In contrast to snow algae, they have evolved different adaptation strategies, being able to persist in the harsh ice conditions with relatively thin and less rigid cell walls.14 Another reason could be the lack of representative species in culture collections and our inability so far to grow ice algae in the laboratory.59 However, some information can be concluded from other Zygnemataeceae species that can be found in polar and high-alpine soil crusts with high desiccation stress. For instance, in laboratory experiments, these Zygnemataeceae showed increased production of phenolic compounds under UV exposure67 and nitrogen limitation induced formation of pre-akinetes,68 which showed higher desiccation tolerance than their vegetative stages.69 Therefore, both snow and ice algae seem to depend on a range of pigment and fatty acid production to release them from the light, desiccation and nutrient stress experienced on glaciers and ice sheets.Compared to what we know about snow algae, the dearth of knowledge on ice algae is striking. One reason could be that they have often been overseen within the similarly coloured cryoconite dust on glacial surfaces.21 Therefore, cryoconite holes are a very different type of habitat on the ice surface as the cryoconite (sediment) material is well submerged in water that is just above the freezing point. The main primary producer in cryoconite holes is cyanobacteria and the filamentous cyanobacterium Phormidesmis priestleyi, which is known from Arctic, Alpine and Antarctic environments,15 exhibits adaptations that contribute to the development of cryoconite granules.70The surfaces of glaciers and ice sheets are pockmarked by characteristic pits known as cryoconite holes Fig. . CryoconP. priestleyi that influences interactions with their environment is their ability to release compounds into the environment in the form of extracellular polymeric substances, which in turn include proteins, lipids, polysaccharides and other secondary metabolites. The polysaccharide component (referred to as extracellular polysaccharide) are formed of complex heteropolysaccharides, most of which contain at least six monosaccharides71 and can be either released into the environment or bound to cyanobacterial filaments to form a protective sheath or capsule. Both extracellular polymeric substances and extracellular polysaccharides are denominated collectively as ‘‘EPS’’, but for the purposes of this review, EPS refers only to extracellular polysaccharide. The production of EPS in polar cyanobacteria may serve a number of different purposes that assist in survival under the kind of harsh conditions experienced in glacial habitats. For example, EPS is known to act as a cryoprotectant that prevents the disruption of cell membranes by freezing,72 can act as a site to deposit UV protective compounds, and may assist in scavenging of metal ions and other nutrients.71 In addition to these adaptive properties, EPS can have a substantial influence on the environment and is key to both the formation of cryoconite and stabilisation of microbial biofilms.Perhaps the most fundamental property of filamentous cyanobacteria such as 73 and acts as a glue that binds together inorganic particles, living microorganisms, and other organic matter in a similar manner to that seen in desert environments.74 The overall size of cryoconite granules is determined by the interplay of physical attachment of granules by cyanobacterial filaments, adhesion of particles by EPS, and degradation of organic matter by heterotrophs within and around the granule.75 In desert soils where Microcoleus vaginatus performs a similar function to P. priestleyi, the EPS sheath binds strongly to sand particles;76 similarly, in cryoconite it is bound EPS rather than released polysaccharides that appears to be most important in determining granule size.78 Chemotaxis and motility is also likely to be an important factor in establishing microbe—cryoconite interactions, both in terms of cyanobacterial motility and attachment to cryoconite granules79 and chemotaxis of heterotrophs to cyanobacterial exudates.80 Overall, such activity is instrumental in collecting material on glacier surfaces into cohesive cryoconite holes that would otherwise be dispersed over wide areas.Rather than being distributed throughout the water column, cryoconite granules are found at the bottom of cryoconite holes, with associated biofilms forming surface attached assemblages rather than forming marine snow like aggregates. During the cryoconite granule formation, cyanobacterial EPS contributes to the aggregation of cryoconite granules by promoting bioflocculation81 Cyanobacterial EPS acts as a substrate that allows for the growth of a complex community of heterotrophs resulting in mature biofilms.76 In microbially dominated ecosystems, EPS can represent a major source of biologically labile carbon to fuel the microbial web82 and is both consumed by heterotrophs and re-assimilated by cyanobacteria.83As well as influencing the physical structure of cryoconite, cyanobacteria are also of substantial importance to the food web of cryoconite holes, with between 75% and 95% of available carbon in cryoconite having been attributed to cyanobacterial photosynthesis.85 This low relative abundance of cyanobacteria in cryoconite holes compared to their impact upon the environment highlights their importance as ecosystem engineers and supports their position as keystone species in these environments.86 Most of the cyanobacterial biomass is probably composed of EPS .Although cyanobacteria are clearly important organisms in cryoconite holes, they appear to represent a relatively small fraction of the microbial community in some diversity studies. In a metagenome of an alpine cryoconite ecosystem, Proteobacteria were dominant numerically with 63.3% of reads being attributed to this phylum. A considerable fraction of assembled contigs were taken up by Bacteriodetes (14%) and Actinobacteria (11.3%) while a much smaller number of contigs were attributed to cyanobacteria (2.5%) ref. . Similar87 cryoconite cyanobacteria appear to be psychrotolerant rather than psychrophilic, despite the extreme cold. Optimal rates of carbon fixation by cryoconite cyanobacteria were found to be much higher in warmer conditions than the ambient temperature usually experienced by these organisms.81 Furthermore, the genome of P. priestleyi BC1401 revealed no clear indication of the typical genomic hallmarks of cold adaptation88 and the mechanism to produce the cyanobacterial sunscreen pigment scytonemin89 was absent from the P. priestleyi BC1401 genome. Yet adaptations to a coldlife style are still likely to exist in these organisms. Changes in fatty acid composition in response to low temperature are known in cyanobacteria90 while some cyanobacteria have shown light and temperature acclimation through the production of carotenoids and modification of the photosynthetic apparatus.92 However, it is not yet known the extent to which these mechanisms might be operating in cyanobacteria inhabiting cryoconites. Several pathways responsible for EPS biosynthesis were identified in P. priestleyi BC1401  for remote Arctic, Antarctica or Alpine locations. Most sampling and analyses approaches are copied from clean room technologies that are not necessarily transferrable to sampling air from remote regions.6 A study looking at the bacterial biogeography in cryoconites on various Svalbard glaciers identified a core community  identified in cryoconites) that are present in mean relative abundance >1% per sample.86 Those OTUs are also often found in other cryospheric systems. Cyanobacteria Leptolyngbya and Phormidium were key in Gokul et al.86 study demonstrating their importance as cryoconite engineer species (see section above), while among the heterotrophs, the families Microbacteriaceae and Intrasporangiaceae among the Actinobacteria seem to be key components of cryoconites. Other studies have also identified the genus Polaromonas as abundant and important members of glacial ice in Arctic, Antarctic and Alps.98 The diversity among Polaromonas is large and a comparison of cultures between Arctic and Antarctica shows that, although having similar 16S ribosomal RNA (rRNA) genes, the metabolic traits between the poles are different, suggesting adaptation of the genus to different environmental conditions.98 More metagenomic studies of glaciers and ice sheets will provide more opportunities to link diversity and functionality of these habitats. A recent metagenomic study shows that the diversity of functions in cryoconite holes is comparable to any other environment, with a range of metabolic pathways associated with organic carbon degradation and acquisition of nutrients.84 A metagenome from Alpine and Himalayan glaciers also revealed high abundance of genes associated with heterotrophic anoxygenic phototrophy.97Heterotrophic bacteria are common within the various supraglacial habitats where they play an important role in nutrient cycling from decaying organic matter. Proteobacteria, particularly Alpha- and Beta-Proteobacteria, Bacteroidetes and Actinobacteria dominate the heterotropic communities at the ice surface, yet spatial differences are apparent between different scales. Depending on the spatial scale of comparison, bacterial community composition may differ between snow, ice and cryoconite holes on the same glacier,99 Studies of fungi in snow and ice are rare and mostly limited to snow molds, which are not active in the snow itself, but attack dormant plants under the snow cover.100 Psychrophilic basidomycetous yeasts have been reported in Arctic,101 Antarctic102 and alpine settings.103 Only recently have Chytridiomycota been described as abundant in alpine and Arctic snow,104 where they are believed to play important roles in nutrient release through their saprotrophic or parasitic activities105 and in snow food-web dynamics.104 Nevertheless, their diversity and function in snow are poorly known. Brown et al.51 found patterns of co-occurrence between these Chytridiomycota and snow algae and suggested that they either share similar environmental tolerance or that the algae act as an environmental filter in fungal community assembly.Archaea have also been detected at the surface of glaciers in snow and ice samples, although the diversity is usually very limited, as the number of studies available or the number of sites of which they are found. Usually when Archaea are found at the surface of glaciers, ammonium-oxidising-Archaea are the dominant group.106 Bellas et al.107 calculated that there is strong viral shunt in cryoconite holes where viral production seemed to cause nearly all the bacterial mortality of those habitats. This strong top-down control must impose selective pressures on the bacterial community. This has been recently demonstrated by another study in incubations under ambient temperature and nutrient conditions.108 On the other hand, Rassner et al.108 also demonstrated that addition of nutrients to cryoconite bacterial communities allow for certain members of the community  to thrive and escape its viral control. Analyses of putative viral genomes from Svalbard and Greenland cryoconites reveal a range of novel group of viruses, including groups with unusual life strategies and genes to control the replication of their hosts.109 There is certainly a lot more to learn on how viruses could control the microbiome of glaciers and ice sheets.There a few studies conducted in supraglacial habitats demonstrating that viruses have an important role in controlling bacterial mortality and potentially the release of labile dissolved organic matter to downstream environments.2 production.111 Those conditions, in combination with lack of light and redox potential that ranges from well oxygenated to completely anoxic habitats, provide ideal conditions for the development of a consortia of cold-adapted heterotrophic and chemoautotrophic microbes that can further provide additional reactions to accelerate rock weathering under the ice.112 For instance, Boyd et al.112 detected increased chemolithotrophic activity from Sideroxydans lithotrophicus, an iron sulfide-oxidising autotrophic bacterium commonly found in subglacial environments in association with abiotic pyrite oxidation. Close relatives of other bacterial taxa associated with the sulphur and iron cycle are often found in subglacial systems in both subglacial lakes and under glaciers.115 Examples include genus Thiobacillus and Thiomicrospira, which have been found in subglacial settings in both Arctic and Antarctica.117 Boyd et al.112 also demonstrated that increase in sulphate levels in subglacial meltwater, in turn, was associated with increased calcite and dolomite dissolution of those waters.Under temperate and polythermal glaciers, as well as large sections of the Greenland and Antarctic ice sheets, liquid water can also occur due to a combination of basal ice being at the pressure-melting point and energy coming from geothermal and frictional activities. Those subglacial systems have a high rock:water ratio, which generates high amounts of weathering products and nutrients. Weathering can happen abiotically via glacial comminution of the bedrock, generating reactions such as pyrite oxidation and H119 Microbial diversity under the ice is usually far lower than the diversity found in supraglacial environments. However, the taxa found in subglacial environments quite often match well with their subglacial geochemical environment and are also often close relatives to other organisms found in other cold environments . Although they found that shorter-term incubations usually resulted in heterotrophy, the longer term incubations showed clear net autotrophy.Many recent studies have attempted to measure microbial activities in situ on glacier surfaces and have often found significant levels of primary and secondary carbon production,Considering the rates of organic matter production and transformation on glaciers are not trivial, it could be hypothesised that the dissolved organic matter signature from glaciers and ice sheets could carry a strong microbial element . Similar efforts have not yet been fully conducted for the Greenland ice sheet, but comparison of the rates of methane production and molecular data between the Antarctic and Greenland ice sheets indicate that the potential methane storage in Greenland is much lower which is reflected by the different types of organic carbon available beneath each ice sheet.16In situ measurements of microbial activity in subglacial environments are limited relative to measurements at the surface of the ice and quantification of microbial processes in subglacial habitats is inferred from data of microbial diversity, a range of geochemical signatures in runoff water, incubations in laboratory or at ice margin and modelling. A combination of one or more of those approaches can strengthen the assumption that microbes are important players of biogeochemical transformations in subglacial systems.132 Considering that glaciers and ice sheets are simple physical environments formed by very similar mechanisms across the globe, this provides opportunities for studies in microbial population genetics. Lack of molecular data and the restrictions of the morphological classification applied to identify the eukaryotic algae on ice and snow could also impact the conclusion that these organisms seem to have a cosmopolitan distribution.37 At the genus level and most often at the species level snow and ice algal taxa occur in the Arctic, Antarctic and the Alps. Whether the cosmopolitan distribution also extends to the sub-species level has not been sufficiently addressed in previous studies and needs to be included in future work. In the northern hemisphere, it is likely that glaciers had some level of continuity between the high arctic and lower latitude mountains during past glaciations, allowing for genetic exchange between cold environments. Likewise, in longer geological timescales , the whole planet was probably covered by ice.133 The use of molecular markers, comparative genomics in a range of different spatial scales  with close relatives from other non-icy environments, metagenomes and molecular clock information could provide new insights into the origins and evolution of the mechanisms for adaptation in snow and ice algae (Fig. The diversity of the key ecosystem engineers inhabiting glaciers and ice sheets is considered relatively limited. The main primary producers in snow, ice and cryoconite holes seem to be the same across the globe.85 compared to the primary producers. While there is very little doubt about the primary producers fitness to thrive on glaciers and ice sheets, which evidenced by their ability to produce and accumulate organic matter in these habitats, very few studies have determined how much of the bacterial heterotrophic community is actually active in the supraglacial environment. In a recent study, where co-extracted 16S rDNA and rRNA from cryoconite samples collected at the margins and the interior of the Greenland ice sheet were analysed, Stibal et al.134 found very different bulk and active bacterial communities. By comparing the bulk DNA with the potentially active RNA community, Stibal et al.134 provided evidence that, although the gene pool in cryoconites is more diverse at the margins, reflecting the contribution of organisms from adjacent ecosystems to the ice, only a fraction of this community seems to be active. On the other hand, the bacterial community in cryoconites collected ca 30 Km from the margin of the ice seems to be represented by almost entirely of active organisms. Similar studies would certainly help to provide insights about the specific microbial community that is intrinsic to the glacial and ice sheet microbiome. In fact, this is one of the main reasons why small valley glaciers need to be differentiated from ice sheets concerning their microbial processes. The former are much more under the influence of transport of a microbial community and nutrients from adjacent ecosystems and a substantial fraction of the community is probably ill equipped to thrive on the ice. Ice sheets operate on longer time scales, which could allow for more opportunities for cold adaptation to occur.The associated heterotrophic bacterial community is far more diverse and likely influenced by a range of local factors


It can be concluded the IR-1 had broad tropism for respiratory tract, digestive system, and renal tissue, reflecting its epitheliotropic nature, but it caused the most severe lesions in the respiratory tract. This was the first pathogenicity study of Iranian IR-1 IBV. Further knowledge of IBV pathogenesis provides the groundwork to inform more effective prevention practices.Avian infectious bronchitis (IB) is a major cause of economic losses in poultry industry. The IB virus primarily affects respiratory tract, but various strains differ in their tropism for other target organs such as kidney and alimentary tract. The objective of this study was to estimate the pathogenicity of Iranian IBV variant (IR-1), which is limited exclusively to Iran. Specific pathogen free chicks were inoculated intranasally. Sera, fecal swabs and different tissue samples were collected on different days post infection (DPI). Clinical signs, gross pathology and histological changes were recorded. The viral load was quantified in the RNA extractions from different tissue samples using real-time PCR. Anti-IBV antibodies were detected in serum samples. The IgG antibody were found on 21 and 28 DPI. Severe histological lesions were observed in the trachea and lung while the lesions in kidney were appeared to be milder. Viral RNA was detected in all tested tissues from 1 DPI to the last day of the experiment. The highest viral load was measured in the trachea and feces on 1 Gammacoronavirus within the Coronaviridae family.1,2 The disease is characterized by respiratory-specific symptoms including gasping, coughing, sneezing, tracheal rales and nasal discharge.3 Chicks of all ages are susceptible to infection and the virus replicates in many tissues including the trachea, lung, spleen, kidney and ovaries. It is recognized that IBV isolates differ in virulence and tissue tropism.4 Most IBV strains are respiratory, inducing histological lesions in the trachea.5 However, Winterfield and Albassam noted that various passage levels of an IB vaccine virus, the Holland strain, were capable of causing microscopic lesions of interstitial nephritis.6 In addition, nephropathogenic strains cause histological lesions in trachea and kidneys.7-9 The first isolation of IBV from Iranian chick flocks was reported in 1994. Later, several Iranian researchers identified the 793/B serotype. This serotype turned out to be one of the predominant types of IBV in Iran.10 Different vaccines (Mass and 793/B vaccines) are now widely used in Iranian poultry farms, but IB outbreaks are still occurring. Recently, one of IBV variants named as IR-1, which was characterized based on partial S1 gene sequencing, has been isolated from Iranian broiler farms. Hashemzadeh et al. investigated the phylogenetic relation-ship of Iranian IBV isolates during 2010 to 2011. Moreover, they identified a new IBV variant, which was not related to any identified genotypes of IBV.11 This new IBV was again detected in a molecular surveillance performed during 2010 to 2014 and designated IR-1.12 Thereafter, IR-1 was isolated from the tissue samples collected from Iranian broiler farms during 2014 to 2015.13 The IR-1 which has been detected in 2010, is only circulated in Iranian poultry farms. In addition, there is no information about IR-1 pathogenic aspects. The aim of the present study was to evaluate IR-1 pathogenicity and tissue tropism in experimentally infected specific pathogen free (SPF) chicks. Infectious bronchitis (IB) is a globally distributed avian disease that represents one of the most persistent sanitary problems in the commercial poultry industry. The etiologic agent, infectious bronchitis virus (IBV), belongs to the genus Strain history and virus titration. The IR-1 genotype of IBV (accession number: KT583580), isolated from Iranian broiler farms in 2014 was used for examining its pathogenicity. The Reed-Muench method14 was used to calculate 50% egg infective dose (EID50) and the isolate was used in the experimental pathogenicity study.Experimental design. A total number of 49 SPF 1-day-old chicks were divided randomly into two groups, 35 chicks in the experimental and 14 chicks in the control group, Each group was housed in a separate isolator. At the age of 14 days, birds in the experimental group were challenged intranasally with the allantoic fluid of IR-1 containing 104 EID50 per 0.10 mL of the virus. Up to 28 days post infection (DPI) chicks were observed and clinical signs and mortality were recorded. Clinical signs were scored as follow: 0 = no abnormal breathing; 1 = slight or rare tracheal rales can be heard after provoking the chick by moving; 2 = moderate or frequent tracheal rales can be heard without moving the chick but not continuously and 3 = marked, continuous tracheal rales can be heard. On 1, 3, 5, 7, 14, 21, and 28 DPI, five chicks from the experimental group and two chicks from control group were randomly selected, the serum was collected for an IB ELISA test. On days 1, 3, 5, 7, 14 and 21, tissue samples of the trachea, lung, spleen, kidneys, proventriculus, cecal tonsils and cloacal swabs were aseptically collected for virus quantification using real-time PCR.Detection of anti-IBV antibodies. The IBV antibody test kit, ProFLOK IBV ELISA kit , was used for the detection of a specific antibody to IBV in the serum samples according to manufacturer’s instruction. Biochemical analysis. The level of uric acid, creatinine, alanine amino transferase activity and aspartate amino transferase activity in the serum samples were measured using standard auto analyzer Selectra ProXSwith software . Primers and probes. Real-time PCR was used to amplify a conserved sequence within the 5′-untranslated region (UTR) of IBV genome and 28s rRNA was used as a reference gene. To amplify a 143-bp fragment of the 5′-UTR of the IBV genome, we used a downstream primer , an upstream primer  and Taqman® dual-labeled probe  which were designated previously.15 For the 28s rRNA gene, the forward primer (28s F 5′GGCGAAGCCAGA GGAAACT-3′), reverse primer (28s R 5′GACGACCGATTTGC ACGTC-3′) and probe (FAM-AGGACCGCTACGGACCTCCACCA-TAMRA) were used to amplify a 61 bp fragments of the gene.16The IBV quantitation standards. The 5' UTR of M41 reference IBV strain and 28s rRNA of chick tissue were amplified using the same primers applied in real-time PCR reaction. The PCR products were examined using agarose-gel electrophoresis. After ethidium bromide staining, the bands were visible only at the expected molecular weights (a 143-bp fragment for 5′-UTR of M41 and 61 bp fragment for 28srRNA). The PCR products for 5′-UTR and 28s rRNA were cloned in a pTG19-T vector and transformed in Escherichia Coli TOP10 competent cells. Plasmid isolation kit mini-preparation  was used to extract plasmids. Before use, the plasmid concentration was determined by spectro-photometry at 260 nm and calculated as genome equivalents (copies) per mL as the molecular weight of the plasmid was known. Serial dilutions were performed to give a final concentration between 102 and 105 (for 5′-UTR) or 102 and 105 (for 28s rRNA) copies for generating the standard curves. The RNA extraction and cDNA synthesis. The RNA was extracted from tissue samples using the Cinna Pure RNA  kit following instructions and treated with Dnaes I. The cDNA was generated using RevertAid First Strand cDNA Synthesis kit .Real-time PCR. The 20 μL real-time PCR reaction contained 2 μL 10X PCR buffer (SinaClon), 1 μL dNTP mix , 0.80 μL Mgcl2 , 0.20 μL CinnaGen Taq DNA polymerase (1 U) (SinaClon), 5 μL template cDNA, primers at a final concentration of 0.10 μM, probe at a final concentration of 0.10 μM and nuclease free water. The reaction was conducted in Corbett Life Science Rotor-Gene 6000 Cycler . The PCR cycling parameters were 95 ˚C for 2 min, then 40 cycles of 95 ˚C for 15 sec and 60 ˚C for 1 min.Histopathological examination. Specimens from the trachea, lung and kidney were placed in 10% buffered formalin, sectioned, stained with hematoxylin and eosin (H & E) and evaluated for histological lesions.Clinical signs, gross lesion and mortality. No clinical signs of respiratory infection were observed until 3 DPI (Table 1). Beginning at 5 DPI, the clinical disease was visible in all infected chicks, most severe on 14 DPI. Chicks in the infected group showed a small amount of catarrhal exudate in the trachea with slight mucosal hyperemia beginning at 3 DPI. Pale and swollen kidneys were observed on 5 to 14 DPI. No clinical signs or gross lesions were observed in the control group of chicks. No mortality occurred in either group.Anti-IBV antibody. Antibody levels against IBV were measured in the serum from the infected or control chicks. Sera from the control group were negative for antibodies against IBV in all samples. In the infected group, no antibodies were detected on 1, 3, 5, 7 and 14 DPI, but anti-body titers on 21 and 28 DPI were 1146.50 and 1486.40, respectively. Furthermore, these values were significantly different from those of the controls (p < 0.05), (< 0.05), .Serum biochemistry. Serum biochemical factors of the IBV infected group did not differ statistically with those of the control group  infected animals. In addition, no obvious lesions were found in the kidney of the H120, an IBV strain with respiratory pathogenicity, infected group.18Histological results showed degenerative changes in the trachea and lungs, but lesions in the kidney were less severe, and there were no signs of epithelial degeneration. Since degenerative changes of renal epithelial cells are almost observed in nephropathogenic IBVs infections,7,8 In histological studies of Benyeda et al., severe lesions were found in the trachea. Lesions induced by the Greek QX and 793/B were less severe. All chicks received the QX, produced more severe kidney lesions than those infected with either M41 or 793/B.19 Our results were in agreement with Benyeda et al. who observed tracheal lesions in all infected groups but kidney lesions were less severe in M41 and 793/B infected chicks. Severe histological lesions of the trachea can occur in the nephropathogenic or QX IBV infections.In general, the clinical signs and histological analysis were in accordance with the results of real-time PCR. The peak of virus replication was recorded until 5 DPI. Thereafter, the signs of disease began to appear and histological changes were occurred gradually. Severe infection was detected during 5 to 14 DPI. Moreover, from 21 DPI, the rising anti-IBV antibody levels reduced the disease severity. There were some factors, which made the real-time assay less reliable in our study of pathogenicity: 1) the epitheliotropic nature of IBV, which caused broad tissue distribution and different genotypes, could replicate in the epithelial cells of respiratory tract, digestive system and renal tissue; 2) the highest load of IBVs could be detected in different organs and various time points after infection and IBVs with similar pathogenic traits might show different trends in real-time PCR and 3) high replication of virus in a distinct organ did not necessarily mean that the virus caused the most severe damage in that particular organ. Real-time PCR results demonstrated that the IR-1 tropism for the respiratory tract, digestive system and renal tissue was due to the epitheliotropic nature of IBV. et al. showed that the histopathology of the tracheas from birds exposed to the virulent and avirulent forms of IBV was markedly different while the virus isolation results were identical in both infected group.24In another pathogenicity study, Geilhausen  It could be concluded that histological observations were more reliable and sensitive criteria in pathogenicity studies. Considering the severe histological changes of the trachea and lung, we estimated that IR-1 mainly affected the respiratory tract. In addition, the virus caused milder histological changes in the kidney, but these changes were not strong enough to alter the renal function. This was the first pathogenicity study of IR-1, which provided new insights on IBV pathogenesis. Middle East countries and Iran have intense trading and uncontrolled movement of inhabitants and animals across borders, which can facilitate the spread of the IR-1 to neighboring countries such as Turkey and Iraq. It is possible that this isolate exists in the neighboring countries, however, to date, it has not been identified. The complete genome sequence of IR-1 is needed to identify the origin and possible genetic recombination.


Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE‐mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state‐of‐the‐art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE‐mediated tumour antigen cross‐presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.  It has been recognized that tumours manipulate immune responses. On the other hand, the overall immune competence of the host could critically determine immune surveillance against cancer and the clinical course. Allergy and atopy are characterized by a systemic bias to Th2 immunity, which may exert a potential influence on cancer development. In fact, allergy and oncology may represent two opposite concepts: whereas immune tolerance is desired in allergy, it is detrimental in cancer. Hence, the establishment of a Task Force on AllergoOncology (AO) within the Immunology Section of EAACI is timely and appropriate. The aim of the Task Force is to connect basic scientists interested in Th2 immunity and cancer with clinical oncologists and to support an interdisciplinary exchange to advance knowledge and understanding of immune responses in both fields. At present, this is the first AO platform worldwide.Previous AO activities have included a first concerted paper A Pre‐Task Force Meeting was held at the EAACI annual conference in Copenhagen in 2014, leading to the establishment of the Task Force within the Interest Group of Immunology, with its first business meeting in Barcelona in 2015. The primary objectives of the Task Force were confirmed: to serve as an interface between the disciplines of oncology and allergy, covering: (i) basic, (ii) translational, (iii) epidemiological and (iv) clinical research, including allergy problems in clinical oncology, as well as (v) mechanisms of tumour‐induced immune modulation and (vi) novel vaccination and immunotherapy approaches harnessing IgE functions to target cancer.The goal of this position paper was to provide an update on developments in the AO field since 2008 The different topics were drafted by subgroups of the Task Force and further discussed, developed and compiled during a meeting in Vienna in 2015. The position paper was thereafter recirculated and critically appraised, and the final version was approved by all Task Force members.FCER1A, IL10, ADAM33, NOS1 and IL4R genes and glioma risk reported in one recent study which requires further replication The epidemiologic association between allergy and cancer risk has been summarized in meta‐analyses, with inverse associations reported for several cancers including glioma, pancreatic cancer, and childhood leukaemia Further research in large‐scale prospective studies using validated measures of self‐reported allergy history and/or biomarkers of allergy is needed, including repeated evaluations over time, sufficient latency with respect to the developing tumour, and detailed data on potentially confounding variables in vivoAlthough studied for decades, our understanding of different immunoglobulin classes in cancer biology is still limited. IgG antibodies are the predominant antibody class for passive immunotherapy. Recent findings elucidated that the tumour microenvironment may specifically promote less potent immunoglobulin isotypes such as IgG4 A preliminary study has reported that both IgE and IgG4 specific towards two of three cancer antigens are elevated in patients with cancer compared with healthy volunteers The enzyme cytidine deaminase (AID) which is responsible for converting cytidine to uracil and thereby induces targeted damage to DNA, is a key driver of immunoglobulin (Ig) somatic hypermutation events and class switch recombination processes that give rise to IgG, IgA or IgE. On the other hand, AID has multifaceted functions linking immunity, inflammation and cancer AID is thought to be expressed predominantly by germinal centre (GC) B cells within secondary lymphoid organs. However, studies on local autoimmunity, transplant rejection, and tissues exposed to chronic inflammation point to the capacity of B lymphocytes to form GC‐like ectopic structures outside of secondary lymphoid tissues The high‐affinity receptor FcεRI tetrameric form αβγ2 is expressed on mast cells and basophils. The trimeric form of the high‐affinity receptor FcεRI (αγ2) and the low‐affinity receptor CD23/FcεRII (b form) Fig. A is exprDepending on the nature and distribution of IgE receptors, different functions might be envisaged. Galectin‐3 is well recognized for its contribution to tumour progression and metastasis development It is important to note that cell surface‐expressed IgE‐binding structures may have different effector functions compared with their secreted forms such as soluble FcεRIα chain Mast cells are perhaps the most classical effector cells of IgE Fig. B. Their A wealth of evidence from human cancers and mouse models of cancer indicates that mast cells via the action of histamine on H1, H2 and H4 receptors contribute to tumour invasion and angiogenesis W‐sh/W‐sh) mice, it was shown that mast cells (and mast cell‐derived MMP‐9) are necessary and sufficient to promote growth of subcutaneously engrafted prostate adenocarcinoma cells Mast cells are attracted to the tumour microenvironment by stem cell factor (SCF) secreted by tumour cells, and secrete pro‐angiogenic factors as well as matrix metalloproteinases (MMPs), which promote tumour vascularization and invasiveness. Stem cell factor is the ligand for CD117 (c‐kit receptor), highly expressed by mast cells. SCF is essential in mast cell recruitment, tumour‐associated inflammation, remodelling and immunosuppression in vivoFibroblast growth factor‐2 (FGF‐2) and VEGF derived from mast cells trigger intense angiogenic responses in vitro when incubated with an anti‐CD20 IgE antibody There is some evidence, however, that these pro‐tumoral activities of mast cells may be subverted by targeting these cells to promote tumour destruction. In a mouse allograft model, triggering of degranulation of mast cells by IgE antibody cross‐linking of cell surface FcεRI resulted in Treg cell impairment and acute CD4+ and CD8+ T cell‐mediated tissue destruction in vitro, implying mechanisms involving innate receptors  and mediators such as alpha‐defensins, TNF‐α, granzyme A and IL‐18 Blood and tissue eosinophilia are prominent features of allergy and also found to be associated with various cancers. Tumour‐associated eosinophilia (TATE) has been reported to correlate with good or with bad prognosis. Epidemiological and clinical studies suggest evidence of intratumoral eosinophil degranulation and tumoricidal activity lowMHCIIhi M1‐like phenotype; in hypoxic areas TAMs show a CD206hiMHCIIlow M2‐like phenotype hiMHCIIlow TAMs Tumour‐associated macrophages (TAMs) differentiate from monocyte precursors circulating in blood Fig. B and areNew strategies aim to re‐educate TAMs to exert antitumour functions. In fact, even in a Th2‐M2 tumour microenvironment macrophages stimulated by IL‐4 and IL‐13 were able to inhibit proliferation of B16‐F1 melanoma cells in vivo. However, IL‐4, a signature Th2 cytokine, efficiently blunted IgE‐mediated cross‐presentation indicative for a feedback mechanism that prevents overshooting CTL responses during allergy Antigen cross‐presentation by DCs is key feature of antitumour immunity as it results in the generation of cytotoxic CD8+ T lymphocytes (CTLs) against tumour antigens. Recently, an IgE‐mediated cross‐presentation pathway has been discovered CD8+ T lymphocytes and Th1 cells play the central role in elimination of tumour cells by the immune system Fig. . Th1 celneu IgE antibody, complexed with antigen, induced enhanced CD4 and CD8 T‐cell activation in vitro compared to antigen‐complexed IgG1 Further, DCs pulsed with anti‐prostate‐specific antigen (PSA) IgE or with anti‐HER2/Different approaches to induce IgE‐mediated adaptive immunity against cancer have been designed.−/− mice, but not in CD23−/− mice. In parallel, using a humanized hFcεRIα mouse model expressing the human FcεRIα chain, human IgE could exert antitumour adjuvant effects A cellular vaccine based on tumour cells infected with modified vaccinia virus Ankara (MVA) and loaded with IgE conferred protection in mice upon tumour challenge, slower tumour growth and increased survival neu or EGFR, a vaccine may be used across different species Other anticancer vaccine approaches are based on specific tumour antigens and tumour antigenic mimotopes which have shown promise in restricting tumour growth in vitro and in vivo  harness the high affinity of this antibody isotype for its cognate Fcε receptors on tissue‐resident and potentially tumour‐resident immune effector cells and (ii) utilize the properties of IgE to exert immune surveillance in Th2 conditions such as in tumour microenvironments. Recombinant antibody technologies and approaches to recombinant IgE (rIgE) production have advanced significantly with a number of antibodies already engineered and tested N‐glycans including terminal galactose, sialic acid and fucose structures N‐glycosylation of IgE is not thought to have a direct impact on its ability to bind to FcεRI or CD23. The contribution of glycosylation on IgE binding to galectin‐3 and galectin‐9 Also the heavily glycosylated structure of the IgE antibody class has to be considered. IgE has seven glycosylation sites, six of which are occupied mainly by complex in vitroEosinophil, monocyte and macrophage‐mediated ADCC/ADCP, antigen presentation by DCs and degranulation of mast cells and basophils, have been identified as potent mechanisms of IgE‐mediated anticancer functions neu and anti‐EGFR IgG1 antibodies, trastuzumab  and cetuximab , respectively, have been cloned and engineered recombinantly as humanized and chimaeric IgE antibodies in vitro using an anti‐human CD20 IgE antibody against OCl‐Ly8 lymphoma cells in vitro, it is important to consider these results in the patient context The ADCC/ADCP functions against cancer of the anti‐folate receptor‐alpha (FRα) IgE, MOv18, have been previously described Furthermore, the ability of patient immune effector cells to eradicate malignant cells must be evaluated to consider: (i) the impact of treatments as chemotherapy or steroid intake on effector cell functions, (ii) the effect of the tumour microenvironment on IgE receptor expression and on killing properties of these effector cells, and (iii) whether IgE immunotherapy may itself re‐educate effector cells to enhance their antitumour functions in vivo efficacy of IgE antibodies against cancer Various animal models have been successfully employed to study the Direct application of human IgE in tumour bearing mice is not applicable as human IgE does not bind rodent Fcε receptors Table . Next, hneu (C6MH3‐B1 IgE) IgE tested in this model significantly prolonged the survival of immunocompetent mice bearing HER2/neu‐expressing tumours Despite these differences, a xenograft model with severe combined immunodeficient (SCID) mice, and a patient‐derived xenograft model of ovarian carcinoma both reconstituted with human peripheral blood mononuclear cells, were successfully used to demonstrate the superior tumour‐killing potential of MOv18 IgE over IgG1 via both CD23 and FcεRI Macaca fascicularis) and rhesus (Macaca mulatta) monkeys, as they have been shown to mediate anaphylaxis induced by human IgE neu (C6MH3‐B1 IgE), administered systemically, was also well tolerated in cynomolgus monkeys Relevant models to address toxicity of human IgE antibodies are nonhuman primates (NHP) such as cynomolgus (Canis lupus familiaris) is another potential model, as dogs suffer from spontaneous cancer and atopic diseases, making them a relevant clinical experimental model neu and canine EGFR expression are highly homologous to the human molecules, and can be targeted by trastuzumab and cetuximab The dog  Therefore, IgE‐ and non‐IgE‐mediated chemotherapy hypersensitivity reactions can be managed by RDD, enabling sensitized patients to receive the full treatment safely, thus representing an important advance in the patient's treatment and prognosis.This position paper summarizes current knowledge and developments in the field of AO since in vivo models relevant to IgE biology have been designed with careful consideration of species‐specific IgE receptor expression profile. Mouse models have been used most often, whereas rats, dogs and NHP may offer new alternatives to address specific questions of potency, safety and function. A couple of recombinant anticancer IgE antibodies are in the pipeline, and parallel interrogation of the same antibody immunotherapies in clinical oncology will determine the predictive value of in vivo models.Emerging studies further support the study of the prototypic Th2 isotype, IgE as a means to combat tumours when directed against cancer antigens through promoting the interaction between effector and cancer cells, and stimulating CTLs via antigen cross‐presentation. Collectively, these findings support the unique properties of IgE to activate anticancer immune responses in passive and active immunotherapy of cancer and provide evidence of safety. Whereas cell‐fixed tumour antigen can trigger cross‐linking of IgE on its Fc receptors expressed on effector cells, monovalent soluble antigen does not. Some Recent findings shed light into the alternative Th2 antibody isotype IgG4 and its expression and functions in melanoma and other cancers In summary, the AO field represents an open interdisciplinary science forum where different aspects of the interface between allergy and cancer are systematically addressed and discussed, gaining thereby previously unappreciated insights for cancer immunotherapy.In situ expression of AID and potential insights into antibody isotype expression in cancer’ section. Untersmayr E contributed to the ‘IgE receptor expression on immune cells and epithelial cells’ section. The following authors contributed to the ‘Effector cells in allergy and cancer’ section: Levi‐Schaffer F, Mekori Y, Bax HJ and Josephs DH; Capron M and Gatault S to ‘Eosinophils’; Bianchini R and Jensen‐Jarolim E to ‘Macrophages’; Fiebiger E to ‘Dendritic cells’; and Janda J to ‘T lymphocytes’. The following authors contributed to the ‘Translational strategies to target cancer’ section: Nigro EA, Vangelista L, Siccardi AG and Jensen‐Jarolim E contributed to the ‘Tumour vaccines and adjuvants’ section; Daniels‐Wells TR, Penichet ML, Spillner E and Gould HJ to ‘Recombinant IgE anticancer antibodies’; Josephs DH, Bax HJ, Karagiannis P, Saul L and Karagiannis SN to ‘In vitro effector functions of IgE antibodies in the cancer context’; Dombrowicz D, Fazekas J, Bax HJ, Daniels‐Wells TR, Penichet ML, Jensen‐Jarolim E and Karagiannis SN to ‘In vivo models in AllergoOncology’; and Castells M, Corrigan C and Spicer JF to ‘Allergy in clinical oncology’. Jensen‐Jarolim E and Karagiannis SN wrote abstract, introduction and conclusion, contributed to several sections and compiled the whole opus; and Fazekas J designed all figures. All authors have read and approved the position paper.Turner MC contributed to the ‘Epidemiology’ section. Singer J, Redegeld F, Spillner E and Karagiannis P contributed to the ‘Th2‐associated antibodies in cancer’ section. Meshcheryakova A, Mechtcheriakova D and Mungenast F contributed to the ‘The authors declare that they have no conflicts of interest.


This elevation was suppressively controlled by targeting Ser129-phosphorylated α-synuclein to the proteasome pathway. Rotenone-induced insoluble α-synuclein was also targeted by Ser129-phosphoryation to the proteasome pathway. Experiments with epoxomicin and chloroquine showed that proteasomal targeting of insoluble Ser129-phosphorylated α-synuclein was enhanced under lysosome inhibition and it reduced accumulation of insoluble total α-synuclein. However, in a rat AAV-mediated α-synuclein overexpression model, there was no difference in the number of total α-synuclein aggregates between A53T mutant and A53T plus S129A double mutant α-synuclein, although Ser129-phosphorylated α-synuclein-positive aggregates were increased in rats expressing A53T α-synuclein. These findings suggest that Ser129-phosphorylation occurs against stress conditions, which increases influx of extracellular Ca2+, and it prevents accumulation of insoluble α-synuclein by evoking proteasomal clearance complementary to lysosomal one. However, Ser129-phosphorylation may provide an ineffective signal for degradation-resistant aggregates, causing extensive phosphorylation in aggregates.Parkinson’s disease (PD) is characterized neuropathologically by intracellular aggregates of fibrillar α-synuclein, termed Lewy bodies (LBs). Approximately 90% of α-synuclein deposited as LBs is phosphorylated at Ser129 in brains with PD. In contrast, only 4% of total α-synuclein is phosphorylated at Ser129 in brains with normal individuals. It is unclear why extensive phosphorylation occurs in the pathological process of PD. To address this issue, we investigated a mechanism and role of Ser129-phosphorylation in regulating accumulation of α-synuclein. In CHO cells, the levels of Ser129-phosphorylated soluble α-synuclein were maintained constantly to those of total α-synuclein in intracellular and extracellular spaces. In SH-SY5Y cells and rat primary cortical neurons, mitochondrial impairment by rotenone or MPPThe online version of this article (doi:10.1186/s40478-017-0452-6) contains supplementary material, which is available to authorized users. Drosophila model of PD, co-expression of α-syn and Drosophila G-protein-coupled receptor kinase 2 (Gprk2) was shown to generate Ser129-phosphorylated α-syn and enhance α-syn toxicity ), sonicated at 30 W for 1 s 5 times, and kept on ice for 30 min. After centrifugation at 12,000×g for 30 min, the resulting supernatant was collected and stored at −80 °C until use. Protein concentrations were measured by the BCA assay (Thermo Fisher Scientific).For preparation of cell lysates, cultured cells were suspended in buffer A , cells were plated onto 10 cm dishes. When cells were 90% confluent, we exchanged the growth media with 6 mL of Opti-MEM (Thermo Fisher Scientific). After further 24 h incubation, CM was collected and centrifuged at 6,000×g for 5 min to remove cell debris. Immediately, 6 mL of CM was added with 1/4 volume of 100% trichloroacetic acid (TCA), incubated for 30 min on ice, and centrifuged at 14,000×g for 5 min. The resultant pellet was washed three times with 300 μL of cold acetone, air dried, and dissolved in 100 μL of Laemmli’s sample buffer containing 2.5% β-mercaptoethanol.For analyzing the levels of insoluble α-syn, 1% Triton X-100 cell lysates were separated by centrifugation at 100,000×g for 30 min . Supernav/v) Tween 20 (TBS-T) for 10 min 3 times. The membrane was blocked by TBS-T containing 5% skim milk for 30 min, incubated in TBS-T containing 2.5% skim milk and primary antibody overnight in the cold room, and further incubated in the same buffer containing the corresponding secondary antibody overnight in the cold room. When we detected phosphorylated α-syn, 50 mM NaF was added to TBS-T containing skim milk. To visualize the signal, membranes were treated with ECL plus (Thermo Fisher Scientific) for detection of total α-syn, including non-phosphorylated and Ser129-phosphorylated forms, and phosphorylated α-syn. Other proteins were detected by supersignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). Signals were recorded using a CCD camera, VersaDog 5000 (Bio-Rad). Levels of total α-syn and Ser129-phosphorylated α-syn were estimated by measuring band intensities with Quantity One software (Bio-Rad). For more quantitative estimation of the expression levels of total α-syn and Ser129-phosphorylated α-syn, we used purified recombinant α-syn proteins and Ser129-phosphorylated α-syn proteins as standards [Samples were denatured at 95 °C for 5 min in Laemmli’s sample buffer containing 2.5% β- mercaptoethanol. Equal protein amounts of denatured samples were subjected to SDS-PAGE on 13.5% polyacrylamide gels and then transferred to PVDF membranes . The transferred membrane was incubated in phosphate-buffered saline (PBS)  containing 4% paraformaldehyde (PFA) with 0.1% glutaraldehyde for detecting Ser129-phosphorylated α-syn for 30 to 60 min or without glutaraldehyde for detecting other proteins as described previously . After itandards , 12. A sThe experiments using rats had been approved by the Animal Subjects Committee of Yamagata University . We usedP values <0.05 were considered statistically significant.We performed each experiment at least three times. Data are expressed as mean ± standard deviation and n represents the number of samples. Homogeneity of variances was checked with Levene’s test. If the variances were homogenous, comparisons were performed by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. In case of nonhomogeneity of variances, comparisons were performed by Welch-ANOVA with Games-Howell post hoc test . The authors declare that the main data supporting the findings of this study are available within the article and Additional files n = 12, r2 = 0.885) , although endogenous α-syn was undetectable  Fig. . The samble Fig. . These f2+ on Ser129-phosphorylation of α-syn, we incubated SH-SY5Y cell lines, which stably expressed wild-type α-syn (wt-aS/SH #4) [P = 0.033; 3.40 ± 0.45-fold increase at 8-h incubation, P = 0.001, n = 5, each group), compared with vehicle control cells     was significantly blocked by the addition of CaM inhibitor W-7 from 0.05 μM  and different CaM inhibitor calmidazolium chloride from 5.0 μM , compared with vehicle control cells  , for 8 hlls Fig. . Cells wup) Fig. . Howeverup) Fig. . The A23up) Fig. . Additioup) Fig. . EGTA anlls Fig. . These fP = 0.013, n = 5), compared with vehicle control cells , and A23187-mediated Ser129-phosphorylation of α-syn (3.30 ± 0.28-fold increase) was significantly inhibited by BAPTA-AM   , compared with vehicle control cells   or 5 μM of rotenone  in a dose-dependent manner  Fig. . After iner Fig. . Total αone Fig. .Fig. 3Ef+ in the presence or absence of Ca2+ chelators. MPP+-mediated Ser129-phosphorylation of α-syn  was significantly inhibited by BAPTA-AM   was significantly inhibited by EGTA    was significantly inhibited by EGTA   was inhibited by W-7    Fig. . To asseup) Fig. . Similarup) Fig. . Rotenonup) Fig. . Additioup) Fig. . Ser129-up) Fig. . These fP < 0.001; 1.68 ± 0.09-fold increase at 10 nM, P = 0.036, n = 4, each group), compared with vehicle control cells . The rotenone-mediated Ser129-phosphorylation of α-syn (1.54 ± 0.11-fold increase) was significantly inhibited by 0.5 mM EGTA   -chase experiments in wt-aS/SH cells. As previously reported, the levels of Ser129-phosphorylated α-syn rapidly decreased Fig. . When ceP = 0.011, n = 4) as compared with vehicle control cells   for 5 days. The cells were then separated into 1% Triton X-100-soluble and 1% Triton X-100-insoluble fractions by centrifugation at 100,000×g for 30 min. The 1% Triton X-100-insoluble fractions were resolved in a solution containing 8 M urea / 2% SDS. Western blots of 1% Triton X-100-soluble fractions showed that 10 and 50 nM rotenone elevated Ser129-phosphorylated α-syn levels without altering total α-syn levels. When we analyzed 1% Triton X-100-insoluble fractions, 50 nM rotenone generated insoluble total α-syn Fig. . The levlls Fig. . Althoug 4) Fig. . We then 4) Fig. . MG132 s 4) Fig. . However 4) Fig. . These fleft panels), epoxomicin did not affect the levels of 1% Triton X-100-insoluble Ser129-phosphorylated α-syn, but did increase 1% Triton X-100-soluble Ser129-phosphorylated α-syn levels. Additionally, epoxomicin did not affect 1% Triton X-100-insoluble total α-syn levels. Chloroquine treatment failed to induce expression of insoluble Ser129-phosphorylated α-syn, but insoluble total α-syn did accumulate . When cells were co-incubated in media containing epoxomicin and chloroquine, insoluble Ser129-phosphorylated α-syn proteins were generated in conjunction with the accumulation of insoluble total α-syn . These findings showed that proteasomal targeting of insoluble Ser129-phosphorylated α-syn was more activated under lysosome inhibition. To further test the effect of Ser129-phosphorylation on the metabolism of insoluble α-syn proteins, we compared insoluble total α-syn levels between cells expressing wild-type α-syn (wt-aS/SH cells) and Ser129-phosphorylation incompetent S129A mutant α-syn (S129A-aS/SH cells). In the wt-aS/SH cells, epoxomicin and chloroquine treatment yielded insoluble total α-syn  more abundantly than chloroquine single treatment    Fig. . Additio 5) Fig. . S129A-a 5) Fig. . These f3 at 2 weeks (n = 5) and 591.9 ± 34.6/mm3 at 4 weeks . Rats expressing A53T plus S129A double-mutant α-syn had 285.8 ± 103.9/mm3 α-syn aggregates at 2 weeks (n = 4) and 585.4 ± 103.3/mm3 at 4 weeks (n = 3). There was no significant difference in the number of total α-syn aggregates between rats expressing A53T single mutant and A53T plus S129A double-mutant α-syn (P = 1.000). These findings showed that Ser129-phosphorylation had no impact on the accumulation of α-syn aggregates in vivo.To determine whether Ser129-phosphorylation affects the formation of α-syn aggregates in vivo, we quantified the number of α-syn aggregates in the striatum of the rat AAV-mediated α-syn overexpression model. These samples were obtained from our previous study . In rats2+ concentration was a key factor in kinase-modulated Ser129-phosphorylation. In support of this, Ser129-phosphorylation required CaM function, which controls a variety of kinases in a Ca2+-dependent manner [+, Ser129-phosphorylation was enhanced through an increased influx of extracellular Ca2+. However, the CHX-chase experiments showed that rotenone-induced Ser129-phosphorylated α-syn was targeted to the proteasome pathway at the same rate as normally phosphorylated α-syn. It should be noted that in the present experimental condition, ATP-dependent proteasome activity was not lost by mitochondrial impairment. These findings suggested that proteasomal targeting played a role in suppressively controlling Ser129-phosphorylated α-syn levels. Then, we examined the role of Ser129-phosphorylation in insoluble α-syn accumulation. Chronic treatment with a low concentration of rotenone for 5 days induced small amounts of insoluble α-syn. CHX-chase experiments with MG132 showed that insoluble Ser129-phosphorylated α-syn was targeted to the proteasome pathway. This finding suggested that proteasomal targeting also played a role in suppressing accumulation of insoluble Ser129-phosphorylated α-syn.To determine the mechanisms and biological role of Ser129-phosphorylation in α-syn aggregate formation, we first examined the regulation of Ser129-phosphorylation in normal soluble α-syn. Our results showed that α-syn was phosphorylated at Ser129 in proportion to the levels of total α-syn. This phenomenon could be explained by two possibilities: 1) responsible kinases are constitutively active in a substrate-dose dependent manner; or 2) this regulation system is maintained by dephosphorylating or degrading excess amounts of Ser129-phosphorylated α-syn in reaction to total α-syn levels. To further explore these possibilities, we examined how the regulation mechanism of Ser129-phosphorylation was disrupted. Our results showed that the intracellular Cat manner , 22. In The present data raised a question of why increased levels of Ser129-phosphorylated α-syn were not accompanied by alteration in the levels of total α-syn in soluble and insoluble forms. To address the issue, we measured the change in levels of Ser129-phosphorylation and total α-syn under proteasome or lysosome inhibition, because α-syn is known to degrade through the autophagy-lysosome pathway . TreatmeThese findings were consistent with results from a previous yeast study showing that Ser129-phosphorylation and sumoylation push α-syn aggregates into the proteasome pathway and autophagy-lysosome pathway, respectively, and Ser129-phosphorylation rescues autophagy-lysosome clearance of α-syn by promoting proteasomal clearance when sumoylation is impaired . This prThe present data also raised a question as to relationship between effects of Ser129-phosphorylation on proteasomal targeting of soluble and insoluble α-syn proteins and extensive phosphorylation in α-syn aggregates. To address this, we assessed α-syn aggregates in a rat rAAV model expressing A53T α-syn with or without the S129A mutation. The present data showed that Ser129-phosphorylation had no impact on α-syn aggregate accumulation despite extensive phosphorylation. A previous study demonstrated that fibrillar α-syn proteins were phosphorylated by casein kinase (CK) I or CK II, and they were not good substrates for phosphatases in vitro . Because+ enhanced Ser129-phosphorylation through increased influx of extracellular Ca2+, this elevation was suppressively controlled by targeting Ser129-phosphorylated α-syn to the proteasome pathway. This targeting was seen in insoluble α-syn induced by rotenone. Additionally, proteasomal targeting of insoluble Ser129-phosphorylated α-syn was promoted under lysosome inhibition. This complementary action prevented accumulation of insoluble total α-syn. However, in a rat AAV-mediated α-syn overexpression model, Ser129-phosphorylation did not affect α-syn aggregate formation. Taken together, we propose a model that extensive phosphorylation in α-syn aggregates was consequently generated by an ineffective action of Ser129-phosphorylation for removing degradation-resistant α-syn aggregates. We report that a role of Ser129-phosphorylation in regulating α-syn expression levels is associated with extensive phosphorylation in α-syn aggregates. The levels of Ser129-phosphorylated α-syn were suppressively maintained to be constant to those of total α-syn in intracellular and extracellular spaces. Although mitochondrial impairment by rotenone or MPPAdditional file 1: Figure S1.2+ on Ser129-phosphorylation of α-syn in rat primary cortical neurons. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or anti-β-actin (AC-15) antibody. a Effect of A23187 concentrations on Ser129-phosphorylation. Primary cortical neurons were treated with A23187 at the indicated concentrations for 8 h. b, c Effect of extracellular Ca2+ chelator EGTA (b) or intracellular Ca2+ chelator BAPTA-AM (B-AM) (c) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 0.25 μM A23187 with the indicated concentrations of EGTA or BAPTA-AM for 8 h. d Effect of CaM inhibitor W-7 on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 0.25 μM A23187 with the indicated concentrations of W-7 for 8 h. Representative blots are shown. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Graphs show relative ratios to vehicle control cells. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances . (TIFF 2872 kb)Effects of CaAdditional file 2: Figure S2.2+ chelator BAPTA-AM (b) or extracelluar Ca2+ chelator EGTA (c) on rotenone-induced Ser129-phosphorylation. Neurons were incubated in media containing 1 nM rotenone and the indicated concentrations of BAPTA-AM or EGTA for 1 h. d Effect of CaM inhibitor W-7 on rotenone-induced Ser129-phosphorylation. Neurons were incubated in media containing 1 nM rotenone with W-7 at the indicated concentrations for 1 h. Representative blots are shown. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Graphs show relative ratios to vehicle control cells. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal variances . (TIFF 4854 kb)Effects of mitochondria complex I inhibitor rotenone on Ser129-phosphorylation of α-syn in rat primary cortical neurons. Cell lysates (5 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Effect of rotenone concentrations on Ser129-phosphorylation. Primary cortical neurons were incubated in media containing the indicated concentrations of rotenone for 1 h. Vehicle controls were treated with DMSO at the same final concentration. b, c Effect of intracellular Ca


Structural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes. Changes in the expression and genomic distributions of H1 variants during cell differentiation appear to contribute to phenotypic differences between cell types, but few details are known about the roles of individual H1 variants and the significance of their disparate capacities for phosphorylation. In this study, we investigated the dynamics of interphase phosphorylation at specific sites in individual H1 variants during the differentiation of pluripotent NT2 and mouse embryonic stem cells and characterized the kinases involved in regulating specific H1 variant phosphorylations in NT2 and HeLa cells.bona fide substrate for CDK9, a notion that is further supported by the significant colocalization of CDK9 and pS187-H1.4 to gene promoters in reciprocal re-ChIP analyses. Moreover, treating cells with actinomycin D to inhibit transcription and trigger the release of active CDK9/P-TEFb from 7SK snRNA complexes induces the accumulation of pS187-H1.4 at promoters and gene bodies. Notably, the levels of pS187-H1.4 enrichment after actinomycin D treatment or cell differentiation reflect the extent of CDK9 recruitment at the same loci. Remarkably, the global levels of H1.5-S18 and H1.2/H1.5-S173 phosphorylation are not affected by these transcription inhibitor treatments, and selective inhibition of CDK2 does not affect the global levels of phosphorylation at H1.4-S187 or H1.5-S18.Here, we show that the global levels of phosphorylation at H1.5-Ser18 (pS18-H1.5), H1.2/H1.5-Ser173 (pS173-H1.2/5) and H1.4-Ser187 (pS187-H1.4) are regulated differentially during pluripotent cell differentiation. Enrichment of pS187-H1.4 near the transcription start site of pluripotency factor genes in pluripotent cells is markedly reduced upon differentiation, whereas pS187-H1.4 levels at housekeeping genes are largely unaltered. Selective inhibition of CDK7 or CDK9 rapidly diminishes pS187-H1.4 levels globally and its enrichment at housekeeping genes, and similar responses were observed following depletion of CDK9. These data suggest that H1.4-S187 is a Our data provide strong evidence that H1 variant interphase phosphorylation is dynamically regulated in a site-specific and gene-specific fashion during pluripotent cell differentiation, and that enrichment of pS187-H1.4 at genes is positively related to their transcription. H1.4-S187 is likely to be a direct target of CDK9 during interphase, suggesting the possibility that this particular phosphorylation may contribute to the release of paused RNA pol II. In contrast, the other H1 variant phosphorylations we investigated appear to be mediated by distinct kinases and further analyses are needed to determine their functional significance.The online version of this article (doi:10.1186/s13072-017-0135-3) contains supplementary material, which is available to authorized users. The H1 family of linker histones is important chromatin architectural proteins that bind linker DNA and facilitate higher order chromatin folding . Human cSeveral types of post-translational modifications, including phosphorylation, methylation and acetylation, have been identified in H1 variants, with phosphorylation being particularly abundant –13. AnalOur laboratory identified H1.2-S173, H1.4-S172 and H1.4-S187 as the predominant sites of interphase phosphorylation of H1.2 and H1.4 in HeLa S3 cells. Analyses with antisera that are highly specific for H1.4 phosphorylation at S187 (pS187-H1.4) suggest that pS187-H1.4 is enriched at sites of transcription by RNA polymerases I and II , but howRecent evidence suggests that individual H1 variants play significant roles in embryonic stem (ES) cell differentiation. Analyses of the abundance of the mRNAs encoding seven somatic H1 variants suggest that they are differentially expressed during the differentiation of human ES cells (hESCs) in vitro . H1 trip6 cells per T-75 flask or 1.33 × 104 cells/cm2 in DMEM, supplemented with 10% FBS and 10 μM all-trans retinoic acid (RA). mESCs were maintained in DMEM with high glucose, supplemented with 15% FBS , 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM β-mercaptoethanol (Sigma), 2 mM l-glutamine , 1000 U/mL LIF  and 1× penicillin/streptomycin (Gibco) on gelatin-coated plates. The cells were fed with fresh medium daily and subcultured in new gelatin-coated plates by trypsinization every other day. Differentiation was induced by seeding cells in ES cell medium without LIF. HeLa cells were grown in DMEM + 10% FBS and subcultured by trypsinization. Cells were treated with flavopiridol (NIH AIDS Reagent Program), NU-6140 (Tocris), actinomycin D (Fisher), α-amanitin (Cayman), triptolide (Tocris) or THZ1 (ApexBio) dissolved in DMSO to selectively inhibit RNA Pol II or CDK activities as described in the figure legends.NTERA-2/D1 (NT2) human embryonal testicular teratocarcinoma cells and W4/129S6 mESCs were obtained from Dr. Fei Wang (UIUC). NT2 cells were grown in DMEM + 10% FBS and subcultured by scraping. Differentiation was induced by dissociating cells with trypsin, followed by seeding at a density of 1 × 102SO4 as described previously  to buffer B . Fractions were collected by time, and proteins were recovered by precipitation with 20% TCA.Crude histones were extracted from isolated nuclei with 0.4 N Heviously . Crude HsiRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s protocol. Cells were seeded the day before transfection in order to reach 60–80% confluency at the time of transfection. Transfection reagent and siRNA were diluted in Opti-MEM (Gibco), mixed, and incubated for 5 min at room temperature. The complexes were then added directly to cell cultures, and the cells were harvested 72 h later.https://imagej.nih.gov/ij/.Whole-cell lysates or histone extracts were electrophoresed in 15% polyacrylamide gels (6% gel for Pol II blots) containing SDS, transferred to a PVDF membrane and blocked with 5% milk powder in TBS for 1 h at room temperature. The blocked membrane was then incubated with primary antibody at 4 °C overnight, washed with TBST, and incubated with secondary antibody conjugated with HRP  for 1 h at room temperature, washed with TBST again, developed with chemiluminescence reagents  and images recorded with a series of lengthening exposures on X-ray films. Bitmap images were generated from selected films using a flatbed scanner and densitometry performed using ImageJ The pS18-H1.5 antisera were generated by immunizing rabbits with a synthetic phosphopeptide (CPVEK-phosphoserine-PAKK) conjugated to maleimide-activated keyhole limpet hemocyanin (Thermo Fisher Scientific) using standard procedures. Pan antisera to H1.0 and H1.5 were generated by immunizing rabbits with full-length recombinant human H1.0 or H1.5 as described previously . The antFor peptide competition assays, 10 µg of non-phosphorylated (CPVEKSPAKK) or phosphorylated (CPVEKpSPAKK) H1.5-S18 peptides (Genscript) was incubated with 2 µL of primary antisera in a small volume (500 μL) of PBS for 2 h at room temperature. These mixtures were then further diluted in TBST for use in immunoblotting.g for 10 min, the supernatants were diluted tenfold with ChIP dilution buffer. Aliquots representing 1–2 × 106 cells in 1.0 ml final volume were used for each pull down. Samples were incubated with specific antibodies  at 4 °C overnight. Immunocomplexes were incubated with 50 μL BSA-blocked protein G Dynabeads (Invitrogen) for 4 h at 4 °C, collected using a magnetic rack, and washed sequentially with ChIP wash buffer I, II, III and twice with TE. Beads were eluted twice with 200 μL 1% SDS in 0.1 M NaHCO3 at 65 °C for 10 min. The combined eluates were made 200 mM NaCl , incubated at 65 °C overnight to reverse cross-links, digested with 50 μg/ml RNase A at 37 °C for 30 min, and then digested with 50 μg/ml proteinase K at 50 °C for 1 h. The DNA fragments were then purified by phenol/chloroform extraction, recovered by ethanol precipitation using 20 μg glycogen as a carrier, and dissolved in 50 μL of deionized water. For re-ChIP assays, immunoprecipitations from the first ChIP were washed sequentially as described above. The immunocomplexes were eluted with 10 mM DTT in TE at 37 °C for 30 min, diluted 20 times with ChIP dilution buffer and then immunoprecipitated with the second antibody using standard ChIP protocol. ChIP products were quantitated by real-time PCR using SYBR Green master mix (Applied Biosystems) and the primers listed in Additional file Chromatin immunoprecipitation (ChIP) experiments were performed as described previously with minor modifications . Cells wWe have generated a collection of highly specific antisera, raised against synthetic phosphopeptides, which recognize phosphorylation at single sites that are unique to individual human H1 variants or are shared between just two variants. We have also raised “pan” antisera against individual full-length recombinant human H1 variants that specifically recognize the intended variant regardless of whether it is phosphorylated or not. The former provide a relative measure of the levels of phosphorylation at defined sites between samples, whereas the latter can be used to confirm that equivalent amounts of a particular H1 variant, regardless of phosphorylation status, are present in the samples being compared. The specificity of our antisera to pS173-H1.2/5, pS187-H1.4 and pan-H1.4 has been described previously . The speControl analyses revealed that expression of the Oct-4, Sox-2 and Nanog transcription factors all decreased significantly in NT2 cells after RA addition, confirming differentiation and the loss of pluripotency in cultures during the treatment interval Fig. a. SimilaWe used hydrophobic interaction chromatography (HIC) as an antibody-independent approach to analyze crude H1 from pluripotent and differentiated NT2 cells. HIC is a convenient alternative to two-dimensional approaches for monitoring H1 variant modification dynamics that provides partial or complete resolution of H1 variants and their phosphorylated forms . HIC anaSome, but not all, of these changes were observed when mES cells differentiated. In particular, the level of pS18-H1.5 in differentiated mESC was also markedly decreased  activation and lead to decreased levels of pS187-H1.4. Initial experiments revealed that THZ1 reduced global levels of pS187-H1.4 in HeLa cells in a dose- and time-dependent manner  , 76. TheEmbryonic stem cells are pluripotent cells derived from the inner cell mass of the early embryo that are able to self-renew and differentiate into all three germ layers , 79. A rOur data imply that enrichment of pS187-H1.4 at the promoters of pluripotency factor genes in pluripotent cells, and at the promoters of housekeeping genes (and presumably other types of genes) in both pluripotent and differentiated cells, is associated with their transcriptional activity. Interphase H1 phosphorylation has been implicated in facilitating transcription in a gene-specific fashion in several model systems. ChIP analyses with an antisera that recognizes multiple H1 phosphorylations suggest that H1 phosphorylation levels correlate directly with the hormone-inducibility and transcriptional competency of the murine mammary tumor virus (MMTV) promoter , 86. ThiTetrahymena macronuclear H1, which lacks SPXZ motifs [CDK2 has been suggested to be an interphase H1 kinase, but the evidence is equivocal. FRAP analyses suggested that the mobility of GFP-H1.4, but not GFP-M1-5 , was positively correlated with the level of CDK2 activity, and this difference was minimized upon CDK2 inhibition . HoweverZ motifs , to deteZ motifs –92, and Z motifs , 93 and Z motifs , did notMultiple lines of evidence presented here strongly implicate CDK9 as the predominant interphase kinase for H1.4-S187 in NT2 and HeLa cells. Brief (one hour or less) treatments with the preferential CDK9 inhibitor FLVP significantly diminish the global level of pS187-H1.4 in both NT2 cells Fig.  and HeLabona fide interphase kinase for H1.4-S187 since ActD induces the release of active P-TEFb from 7SK snRNA complexes [Given the roles of CDK7 and CDK9 in regulating RNAP II transcription , 57, theomplexes , 94, 95.Our collective data suggest that recruitment of existing active P-TEFb complexes, or P-TEFb released from 7SK complexes, to sites of transcription results in the accumulation and enrichment of pS187-H1.4 that facilitates RNAP II transcription by promoting the transient dissociation of H1.4 or possibly by other mechanisms. A variety of mechanisms have been implicated in releasing P-TEFb from 7SK complexes, including post-translational modification of 7SK snRNP components and direct interactions with RNA binding proteins or transcriptional regulators , 97. TheRecent data suggest a possible mechanism for our observations that the levels of CDK9 and pS187-H1.4 at pluripotency factor gene promoters are reduced by differentiation Figs. , 8b. BRDAn obvious difference that distinguishes the amino acid sequences of metazoan H1 variants from each other is the number and positions of SPXZ sites they possess for interphase phosphorylation. Human H1.1 and H1.3 each have one at the same relative position (S183 or S189). H1.2 also has one, but at a different position (S173). H1.4 has two (S172 and S187) and H1.5 has three . Other human H1 variants either lack SPXZ sites or they occur at non-conserved positions . These dbona fide substrate for CDK9 that is phosphorylated during transcriptional activation, and that reductions in the levels of CDK9 and pS187-H1.4 at pluripotency factor gene promoters contribute to their repression of during cell differentiation. Interphase phosphorylation at other sites in H1 variants appears to be regulated by different kinase pathways, and this is expected to contribute to functional diversity among different H1 variants.In this study, we demonstrated that interphase phosphorylation of H1 variants is reduced preferentially at specific sites during pluripotent cell differentiation and that the enrichment of pS187-H1.4, but not pS18-H1.5, is positively correlated with transcription. Brief inhibition of CDK9 or CDK7 with specific inhibitors, or depletion of CDK9, decreased global and gene-specific levels of pS187-H1.4. Remarkably, inhibiting RNAP II transcription with actinomycin D significantly enhanced co-enrichment of pS187-H1.4 and CDK9 at housekeeping genes. In contrast, the global levels of pS18-H1.5 or pS173-H1.2/5 are not affected by any of the above treatments, suggesting that these interphase phosphorylations are regulated by different mechanisms. Our findings suggest that H1.4-S187 is a Additional file 1: Table S1. List of ChIP-qPCR primers.Additional file 2: Figure S1. Validations of custom H1 antibodies that have not been published previously. A, Recombinant H1 variants were analyzed by immunoblotting with our custom antisera for H1.5 and H1.0. B, PCA-extracted crude H1 from WI-38 VA-13 cells and a mixture of recombinant H1.5 and H1.0 were analyzed by immunoblotting with our custom antisera against pS18-H1.5. C, Antisera against pS18-H1.5 was mock-treated (none) or preadsorbed with pS18-H1.5 antigen peptide (pS18) or the corresponding non-phosphorylated peptide (S18) prior to immunoblotting with HeLa whole-cell lysate. D, HeLa and WI-38 VA-13 whole-cell lysates (WCL) and PCA-extracted crude H1 (PCAS) from WI-38 VA-13 cells were analyzed by immunoblotting with our custom antisera against H1.5 and H1.0.Additional file 3: Figure S2. H1 variant expression and phosphorylation in differentiating NT2 cells. Crude H1 in acid extracts of nuclei isolated from NT2 cells induced to differentiate with 10 μM retinoic acid for 0, 1, 3 or 7 days was fractionated by hydrophobic interaction chromatography (HIC). Eluate absorbance at 214 nm (Y axis) is plotted relative to time (X axis) for equivalent portions of each separation. The relative elution positions of H1.2, H1.3, H1.4 and H1.5 and the phosphorylation stoichiometry of their major interphase forms, as characterized previously [eviously , are indAdditional file 4: Figure S3. pS187-H1.4 is preferentially enriched in active chromatin. A, HeLa cells were treated with DMSO or 500 nM ActD for 1 h and the levels of CDK9 and pS187-H1.4 at the promoters of ACTG1, GAPDH or MYOD1 and one intergenic region were assessed by ChIP-qPCR. B, HeLa cells were treated with DMSO or 1 µM FLVP for 1 h and the levels of CDK9 and pS187-H1.4 at the promoters of ACTG1, GAPDH or MYOD1 and one intergenic region were assessed by ChIP-qPCR. Negative control ChIP assays employed non-immune rabbit IgG (rIg) in place of primary antisera for the first ChIP. The data are expressed as percent relative to input DNA .