A 4 year observation of gastrointestinal nematode egg counts, nemabiomes and the benzimidazole resistance genotypes of Teladorsagia circumcincta on a Scottish sheep farm.

Anthelmintic resistance threatens the sustainability of sheep production globally. Advice regarding strategies to reduce the development of anthelmintic resistance incorporates the outcomes of modelling exercises. Further understanding of gastrointestinal nematode species diversity, and population dynamics and genetics (which may vary between species) is required to refine these models; and field studies combining faecal egg outputs, species composition and resistance genetics are needed to calibrate them. In this study, faecal samples were taken from ewes and lambs on a commercial farm in south-eastern Scotland at approximately 3 t-4 week intervals between spring and autumn over a period of 4 years. Faecal egg counts were performed on these samples, and L3 were collected from pooled coprocultures. Deep amplicon sequencing was used to determine both the species composition of these L3 and the proportions of benzimidazole-resistant single nucleotide polymorphisms in the isotype-1 ß-tubulin locus of the predominant species, Teladorsagia circumcincta L3. Despite consistent management throughout the study, the results show variation in gastrointestinal nematode species composition with time and between age groups, that was potentially associated with weather conditions. The F200Y benzimidazole resistance mutation is close to genetic fixation in the T. circumcincta population on this farm. There was no evidence of variation in isotype-1 ß-tubulin single nucleotide polymorphisms frequency between age groups, and no genetic evidence of reversion to benzimidazole susceptibility, despite targeted benzimidazole usage. This study highlights the need to include speciation when investigating gastrointestinal nematode epidemiology and anthelmintic resistance, and serves as an example of how genetic data may be analysed alongside species diversity and faecal egg counts, when markers for other anthelmintic classes are identified.

CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease).

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child's lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.

Does logging and forest conversion to oil palm agriculture alter functional diversity in a biodiversity hotspot?

Forests in Southeast Asia are rapidly being logged and converted to oil palm. These changes in land-use are known to affect species diversity but consequences for the functional diversity of species assemblages are poorly understood. Environmental filtering of species with similar traits could lead to disproportionate reductions in trait diversity in degraded habitats. Here, we focus on dung beetles, which play a key role in ecosystem processes such as nutrient recycling and seed dispersal. We use morphological and behavioural traits to calculate a variety of functional diversity measures across a gradient of disturbance from primary forest through intensively logged forest to oil palm. Logging caused significant shifts in community composition but had very little effect on functional diversity, even after a repeated timber harvest. These data provide evidence for functional redundancy of dung beetles within primary forest and emphasize the high value of logged forests as refugia for biodiversity. In contrast, conversion of forest to oil palm greatly reduced taxonomic and functional diversity, with a marked decrease in the abundance of nocturnal foragers, a higher proportion of species with small body sizes and the complete loss of telecoprid species (dung-rollers), all indicating a decrease in the functional capacity of dung beetles within plantations. These changes also highlight the vulnerability of community functioning within logged forests in the event of further environmental degradation.

Placing the RPL32 Promoter Upstream of a Second Promoter Results in a Strongly Increased Number of Stably Transfected Mammalian Cell Lines That Display High Protein Expression Levels.

The use of high stringency selection systems commonly results in a strongly diminished number of stably transfected mammalian cell lines. Here we placed twelve different promoters upstream of an adjacent primary promoter and tested whether this might result in an increased number of colonies; this is in the context of a stringent selection system. We found that only the promoter of the human ribosomal protein, RPL32, induced a high number of colonies in CHO-DG44 cells. This phenomenon was observed when the RPL32 promoter was combined with the CMV, SV40, EF1-α, and the ß-actin promoters. In addition, these colonies displayed high protein expression levels. The RPL32 promoter had to be functionally intact, since the deletion of a small region upstream of the transcription start site demolished its positive action. We conclude that adding the RPL32 promoter to an expression cassette in cis may be a powerful tool to augment gene expression levels.

Influence of wing loading on the trade-off between pursuit-diving and flight in common guillemots and razorbills.

Species of bird that use their wings for underwater propulsion are thought to face evolutionary trade-offs between flight and diving, leading to the prediction that species with different wing areas relative to body mass (i.e. different wing loadings) also differ in the relative importance of flight and diving activity during foraging trips. We tested this hypothesis for two similarly sized species of Alcidae (common guillemots and razorbills) by using bird-borne devices to examine three-dimensional foraging behaviour at a single colony. Guillemots have 30% higher wing loading than razorbills and, in keeping with this difference, razorbills spent twice as long in flight as a proportion of trip duration whereas guillemots spent twice as long in diving activity. Razorbills made a large number of short, relatively shallow dives and spent little time in the bottom phase of the dive whereas guillemots made fewer dives but frequently attained depths suggesting that they were near the seabed (ca. 35-70 m). The bottom phase of dives by guillemots was relatively long, indicating that they spent considerable time searching for and pursuing prey. Guillemots also spent a greater proportion of each dive bout underwater and had faster rates of descent, indicating that they were more adept at maximising time for pursuit and capture of prey. These differences in foraging behaviour may partly reflect guillemots feeding their chicks single large prey obtained near the bottom and razorbills feeding their chicks multiple prey from the water column. Nonetheless, our data support the notion that interspecific differences in wing loadings of auks reflect an evolutionary trade-off between aerial and underwater locomotion.

Fine-scale foraging behaviour of a medium-ranging marine predator.

1. Movement patterns of predators should allow them to detect and respond to prey patches at different spatial scales, particularly through the adoption of area-restricted search (ARS) behaviour. Here we use fine-scale movement and activity data combined with first-passage time (FPT) analysis to examine the foraging strategy of northern gannets Morus bassanus in the western North Sea, and to test the following hypotheses: (i) birds adopt a hierarchical foraging strategy characterized by nested ARS behaviour; (ii) the locations and characteristics of ARS zones are strongly influenced by physical oceanography; (iii) the initiation of ARS behaviour is triggered by the detection and pursuit of prey; (iv) ARS behaviour is strongly linked to increased foraging effort, particularly within nested ARS areas. 2. Birds on 13 of 15 foraging trips adopted ARS behaviour at a scale of 9.1 +/- 1.9 km, and birds on 10 of these 13 trips adopted a second, nested ARS scale of 1.5 +/- 0.8 km, supporting hypothesis 1 above. ARS zones were located 117 +/- 55 km from the colony and over half were within 5 km of a tidal mixing front ~50 km offshore, supporting hypothesis 2 above. 3. The initiation of ARS behaviour was usually followed after only a short time interval (typically ~5 min) by the commencement of diving. Gannets do not dive until after they have located prey, and so this pattern strongly suggests that ARS behaviour was triggered by prey detection, supporting hypothesis 3 above. However, ~33% of dives in mixed coastal water and 16% of dives in stratified water were not associated with any detectable ARS behaviour. Hence, while ARS behaviour resulted from the detection and pursuit of prey, encounters with prey species did not inevitably induce ARS behaviour. 4. Following the initiation of ARS behaviour, dive rates were almost four times higher within ARS zones than elsewhere and almost three times higher in zones with nested ARS behaviour than in those without, supporting hypothesis 4 above and suggesting that the foraging success of birds was linked to their ability to match the hierarchical distribution of prey.

Modelling the spatial distribution of ammonia emissions from seabirds in the UK.

Knowledge of the sources and distribution of ammonia (NH3) emissions underpins our understanding of the nitrogen budget. Research has focused on quantifying NH3 emissions from anthropogenic sources, whilst those from natural sources have received little attention internationally. Seabirds excrete large quantities of nitrogen, making seabird colonies a major natural source of NH3. Ammonia emissions from each UK seabird species were estimated and combined with population distribution data to model their spatial distribution. Total NH3 emissions from UK seabirds were estimated at 2.7 kt per year. Seabird emissions are concentrated in remote parts of the UK where anthropogenic emissions are small, so that seabirds often represent the main source of NH3 emissions in these areas. Seabird NH3 emissions were found to have increased by 34% since the 1970s. This corresponds to population changes which may be influenced by human activities, showing that even this natural source can be anthropogenically modified.

Sex-specific foraging behaviour in a monomorphic seabird.

Sexual differences in the foraging behaviour of parents have been observed in a number of sexually sizedimorphic birds, particularly seabirds, and the usual inference has been that these sex-specific differences are mediated primarily by differences in body size. To test this explanation, we compared the foraging behaviour of parents in a monomorphic seabird species, the northern gannet Morus bassanus. Using specially designed instruments and radio telemetry we found that individuals of both sexes were consistent in the directions and durations of their foraging trips. However, there were significant differences in the foraging behaviour of males and females. Female gannets were not only more selective than males in the areas where they foraged, but they also made longer, deeper dives and spent more time on the sea surface than males. As the sexes are morphologically similar in this species, then these differences are unlikely to have been mediated by body size. Our work highlights the need to investigate sexual differences in the foraging behaviour of seabirds and other species more closely, in order to test alternative theories that do not rely on differences in body size.

PH and grain-size variation in leaching tests with bricks made of harbour sediments compared to commercial bricks.

Bricks made of 50% wt. harbour sediments from Bremen, Germany, harbour sediment and nine commercial bricks made of common raw materials were leached in various experiments. The harbour sediment is polluted with heavy metals, e.g. Zn, Cd, Pb and organic compounds, e.g. tributyltin. To assess the environmental impact in the potential use of sediment bricks we consider the influence of pH (4-11) and grain size (50-30 000 microm), the two prime variables in the life-cycle of the bricks. Leachability of trace contaminants increased at acidic pH values and remained low at neutral and alkaline pH values. Leachability increased for smaller grain sizes in relation to the increasing specific surface areas. Grain sizes below 63 microm showed reverse effects for V, Cr, Ni, As, Sr, Mo and Pb due to sorption on the sample material or freshly precipitated phases such as barite, anhydrite or cuprous ferrite. A grain-size fraction of 125-1000 microm was selected in the leaching tests in order to compare different brick types. In general, the leachability of heavy metals from the sediment brick was in the upper range of the commercial bricks. At a temperature of 1050 degrees C thermal treatment of harbour sediments led to an immobilization of most trace contaminants. Chromium, V, As and Mo became even more mobile after thermal treatment, but the enhanced mobilization of V, As and Mo differed strongly among the bricks compared.

Evidence of intra-specific competition for food in a pelagic seabird.

The factors affecting the population dynamics of seabirds have long intrigued biologists. Current data suggest that density-dependent depletion of prey during the breeding season may regulate population size. However, much of the evidence for this has been circumstantial, and the underlying mechanisms are unclear. Here, we show that the per capita population growth rates of northern gannet Morus bassanus at colonies in Britain and Ireland have declined with increasing population size. Furthermore, direct observations reveal that the mean foraging trip duration of breeding gannets is positively correlated with colony size, both among colonies of different sizes in the same year, and within colonies as they change in size. To understand this phenomenon, we have developed a model which demonstrates that disturbance of fish alone can readily generate conditions under which gannets at larger colonies have to travel further to obtain food.

Differential expression of human Polycomb group proteins in various tissues and cell types.

Polycomb group proteins are involved in the maintenance of cellular identity. As multimeric complexes they repress cell type-specific sets of target genes. One model predicts that the composition of Polycomb group complexes determines the specificity for their target genes. To study this hypothesis, we analyzed the expression of Polycomb group genes in various human tissues using Northern blotting and immunohistochemistry. We found that Polycomb group expression varies greatly among tissues and even among specific cell types within a particular tissue. Variations in mRNA expression ranged from expression of all analyzed Polycomb group genes in the heart and testis to no detectable Polycomb group expression at all in bone marrow. Furthermore, each Polycomb group gene was expressed in a different number of tissues. RING1 was expressed in practically all tissues, while HPH1 was expressed in only a few tissues. Also within one tissue the level of Polycomb group expression varied greatly. Cell type-specific Polycomb group expression patterns were observed in thyroid, pancreas, and kidney. Finally, in various developmental stages of fetal kidney, different Polycomb group expression patterns were observed. We conclude that Polycomb group expression can vary depending on the tissue, cell type, and development stage. Polycomb group complexes can only be composed of the Polycomb group proteins that are expressed. This implies that with cell type-specific Polycomb group expression patterns, cell type-specific Polycomb group complexes exist. The fact that there are cell type-specific Polycomb group targets and cell type-specific Polycomb group complexes fits well with the hypothesis that the composition of Polycomb group complexes may determine their target specificity. J. Cell. Biochem. Suppl. 36: 129-143, 2001.

Coexpression of BMI-1 and EZH2 polycomb-group proteins is associated with cycling cells and degree of malignancy in B-cell non-Hodgkin lymphoma.

Polycomb-group (PcG) proteins, such as BMI-1 and EZH2, form multimeric gene-repressing complexes involved in axial patterning, hematopoiesis, and cell cycle regulation. In addition, BMI-1 is involved in experimental lymphomagenesis. Little is known about its role in human lymphomagenesis. Here, BMI-1 and EZH2 expression patterns are analyzed in a variety of B-cell non-Hodgkin lymphomas (B-NHLs), including small lymphocytic lymphoma, follicular lymphoma, large B-cell lymphoma, mantle-cell lymphoma, and Burkitt lymphoma. In contrast to the mutually exclusive pattern of BMI-1 and EZH2 in reactive follicles, the neoplastic cells in B-NHLs of intermediate- and high-grade malignancy showed strong coexpression of BMI-1 and EZH2. This pattern overlapped with the expression of Mib-1/Ki-67, a marker for proliferation. Neoplastic cells in B-NHL of low-grade malignancy were either BMI-1(low)/EZH2(+) (neoplastic centroblasts) or BMI-1(low)EZH2(-) (neoplastic centrocytes). These observations show that low-, intermediate-, and high grade B-NHLs are associated with increased coexpression of the BMI-1 and EZH2 PcG proteins, whose normal expression pattern is mutually exclusive. This expression pattern is probably caused by a failure to down-regulate BMI-1 in dividing neoplastic cells, because BMI-1 expression is absent from normal dividing B cells. These observations are in agreement with findings in studies of Bmi-1 transgenic mice. The extent of BMI-1/EZH2 coexpression correlated with clinical grade and the presence of Mib-1/Ki-67 expression, suggesting that the irregular expression of BMI-1 and EZH2 is an early event in the formation of B-NHL. This points to a role for abnormal PcG expression in human lymphomagenesis. (Blood. 2001;97:3896-3901)

Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation.

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2(+). By contrast, subcapsular immature double-negative (DN) (CD4(-)/CD8(-)) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4(+)/CD8(+)) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2(+) DN and DP thymocytes were dividing, while DN BMI-1(+)/EZH2(-) thymocytes were resting and proliferation was occasionally noted in DN BMI-1(+)/EZH2(+) cells. Maturation of DP cortical thymocytes to single-positive (CD4(+)/CD8(-) or CD8(+)/CD4(-)) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.

The polycomb group protein EED interacts with YY1, and both proteins induce neural tissue in Xenopus embryos.

Polycomb group (PcG) proteins form multimeric protein complexes which are involved in the heritable stable repression of genes. Previously, we identified two distinct human PcG protein complexes. The EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG proteins. Here we show that YY1, a homolog of the Drosophila PcG protein pleiohomeotic (Pho), interacts specificially with the human PcG protein EED but not with proteins of the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins, the interaction between YY1 and EED provides a direct link between the chromatin-associated EED-EZH PcG complex and the DNA of target genes. To study the functional significance of the interaction, we expressed the Xenopus homologs of EED and YY1 in Xenopus embryos. Both Xeed and XYY1 induce an ectopic neural axis but do not induce mesodermal tissues. In contrast, members of the HPC-HPH PcG complex do not induce neural tissue. The exclusive, direct neuralizing activity of both the Xeed and XYY1 proteins underlines the significance of the interaction between the two proteins. Our data also indicate a role for chromatin-associated proteins, such as PcG proteins, in Xenopus neural induction.

Ecology of tropical butterflies in rainforest gaps.

Tropical forest gaps are ephemeral and patchily distributed within forest areas and have very different light environments compared with closed-canopy forest. We used fruit-baited traps to investigate if gaps are exploited by more opportunistic butterfly species compared with closed-canopy forest. Gaps supported a higher diversity of butterflies in terms of species evenness but closed-canopy sites contained species with more restricted geographical distributions. There was little similarity between the assemblages of butterflies trapped in the canopy and those in either gap or closed-canopy sites, but the greater similarity was with gaps, and increased diversity in gaps was partly due to canopy species turning up in gaps. Dispersal rates (as measured by recapture rates) were higher in gaps and there was evidence that butterflies in gaps had relatively larger and broader thoraxes, indicating a flight morphology adapted for faster flight. These results support the notion of a distinctive gap fauna comprising more widespread, mobile species. Habitat modification that opens up the canopy is likely to result in an increase in these widespread species and a decline in understorey species with restricted distributions.

Postnatal development of northern fulmar chicks, Fulmarus glacialis.

The slow growth and large fat stores characteristic of many pelagic seabird chicks were generally assumed to reflect infrequent and unpredictable food provisioning by parents. Much less attention has been focused on the importance of intrinsic physiological processes in shaping patterns of development. In this study, we examined postnatal growth and changes in water content of different organs in fulmar chicks, Fulmarus glacialis, from Fair Isle, United Kingdom. After correcting for body size, mass growth rate was as high as in inshore-feeding species, which did not support the notion of an external constraint on growth imposed by the unpredictability of pelagic prey. Pectoral muscles and plumage grew more rapidly than other tissues. Pectorals also had a high water index, probably indicating slower maturation compared with leg muscles, which need to generate heat earlier on to free adults from brooding requirements. Lean dry mass of liver, kidney, and gut decreased markedly toward fledging, presumably because of high energetic costs of maintaining large metabolic machinery in older chicks and analogous to the situation in adult waders before migration. These results suggest that the general pattern of development of fulmars may be linked to changes in resource allocation as chicks grow and possibly a compromise at the tissue level between cell division and the attainment of mature function.

Coexpression of BMI-1 and EZH2 polycomb group genes in Reed-Sternberg cells of Hodgkin's disease.

The human BMI-1 and EZH2 polycomb group (PcG) proteins are constituents of two distinct complexes of PcG proteins with gene regulatory activity. PcG proteins ensure correct embryonic development by suppressing homeobox genes, and they also contribute to regulation of lymphopoiesis. The two PcG complexes are thought to regulate different target genes and probably have different tissue distributions. Altered expression of PcG genes is linked to transformation in cell lines and induction of tumors in mutant mice, but the role of PcG genes in human cancers is relatively unexplored. Using antisera specific for human PcG proteins, we used immunohistochemistry and immunofluorescence to detect BMI-1 and EZH2 PcG proteins in Reed-Sternberg cells of Hodgkin's disease (HRS). The expression patterns were compared to those in follicular lymphocytes of the lymph node, the normal counterparts of HRS cells. In the germinal center, expression of BMI-1 is restricted to resting Mib-1/Ki-67(-) centrocytes, whereas EZH2 expression is associated with dividing Mib-1/Ki-67(+) centroblasts. By contrast, HRS cells coexpress BMI-1, EZH2, and Mib-1/Ki-67. Because HRS cells are thought to originate from germinal center lymphocytes, these observations suggests that Hodgkin's disease is associated with coexpression of BMI-1 and EZH2 in HRS cells.

Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels.

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.

Cutting edge: polycomb gene expression patterns reflect distinct B cell differentiation stages in human germinal centers.

Polycomb group (Pc-G) proteins regulate homeotic gene expression in Drosophila, mouse, and humans. Mouse Pc-G proteins are also essential for adult hematopoietic development and contribute to cell cycle regulation. We show that human Pc-G expression patterns correlate with different B cell differentiation stages and that they reflect germinal center (GC) architecture. The transition of resting mantle B cells to rapidly dividing Mib-1(Ki-67)+ follicular centroblasts coincides with loss of BMI-1 and RING1 Pc-G protein detection and appearance of ENX and EED Pc-G protein expression. By contrast, differentiation of centroblasts into centrocytes correlates with reappearance of BMI-1/RING1 and loss of ENX/EED and Mib-1 expression. The mutually exclusive expression of ENX/EED and BMI-1/RING1 reflects the differential composition of two distinct Pc-G complexes. The Pc-G expression profiles in various GC B cell differentiation stages suggest a role for Pc-G proteins in GC development.

Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes.

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.