Deficiency of Polη in Saccharomyces cerevisiae reveals the impact of transcription on damage-induced cohesion.

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.

Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability.

Corneal cross-linking (CXL)-A clinical study to evaluate CXL as a treatment in comparison with medical treatment for ulcerative keratitis in horses.

OBJECTIVE: Compare CXL treatment with medical treatment alone in horses with stromal, ulcerative keratitis. ANIMALS STUDIED: 24 horses (24 eyes) with stromal, ulcerative keratitis were included. PROCEDURE: 12 horses were initially treated with CXL, and 12 horses were given conventional medical treatment. Topical medical treatment was added to horses in the CXL group if necessary. Parameters including cytology, microbial growth, time to fluorescein negativity, and time to inhibition of stromal melting were evaluated. RESULTS: After the first day of treatments, a decrease in inflammatory signs and pain from the eye was observed in both groups. Stromal melting ceased within 24 hours regardless of treatment. CXL treatment alone was sufficient in 3 horses with noninfectious, superficial stromal ulcerations. Clinical signs of impaired wound healing were seen after 3-14 days in corneas with suspected or proven bacterial infection treated with CXL only, most likely because of insufficient elimination of bacteria deeper in the corneal stroma or because of re-infection from bacteria in the conjunctiva. The average decrease in stromal ulcer area per day after onset of treatment was almost identical between the groups, and no significant difference in time to fluorescein negativity was found. CONCLUSIONS: We consider CXL a possible useful adjunct treatment of corneal stromal ulcers in horses, especially for melting ulcers and as a potential alternative to prophylactic antibiotic treatment for noninfected stromal ulcers. However, CXL should not be used alone for infected or suspected infected stromal ulcers, because topical antibiotics were required in all horses with proven infectious keratitis.

Age-associated changes in the equine flash visual evoked potential.

OBJECTIVE: To investigate age-associated changes of flash visual evoked potentials (FVEPs) in sedated horses. ANIMAL STUDIED: Twenty-eight clinically healthy Standardbred Warmblooded trotters, aged 36 hours to 28 years. PROCEDURES: Light-adapted FVEPs and FERGs were recorded (An-vision RETIport, Roland-consult, Germany) in response to flash stimuli. Sedation was obtained using alpha-2-agonists intravenously. Akinesia of the eyelids was induced and pupils were dilated. RESULTS: Reproducible FVEPs and FERGs were readily recorded from all foals and horses. The FVEP waveform included up to four positive components (P1-P5) and two negative components (N1 and N2) and FVEP waveform morphology was similar across all age groups. Some differences in peak times and amplitudes associated with increasing age were observed. FVEP amplitudes recorded from newborn foals were well above the amplitudes observed in normal adult horses and FVEP peak times were somewhat shorter. In adult horses, a significant increase in P4 peak time and a gradual decrease in amplitudes, mainly for N2P4, were seen across the life-span. CONCLUSIONS: The overall equine FVEP waveform was similar across the normal life-span of the horse in our cross-sectional study. We found that the visual system of the foal seems to be well developed already at birth. Furthermore, our results showed a decrease in amplitudes and increase in some peak times with increasing age. We recommend that age-matched controls should be used when evaluating foals and young horses in clinical practice, whereas horses over the age of three years can be compared to other adult horses.

Chromosome length influences replication-induced topological stress.

During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

A novel mouse model for the hyper-IgM syndrome: a spontaneous activation-induced cytidine deaminase mutation leading to complete loss of Ig class switching and reduced somatic hypermutation.

We describe a spontaneously derived mouse line that completely failed to induce Ig class switching in vitro and in vivo. The mice inherited abolished IgG serum titers in a recessive manner caused by a spontaneous G → A transition mutation in codon 112 of the aicda gene, leading to an arginine to histidine replacement (AID(R112H)). Ig class switching was completely reconstituted by expressing wild-type AID. Mice homozygous for AID(R112H) had peripheral B cell hyperplasia and large germinal centers in the absence of Ag challenge. Immunization with SRBCs elicited an Ag-specific IgG1 response in wild-type mice, whereas AID(R112H) mice failed to produce IgG1 and had reduced somatic hypermutation. The phenotype recapitulates the human hyper-IgM (HIGM) syndrome that is caused by point mutations in the orthologous gene in humans, and the AID(R112H) mutation is frequently found in HIGM patients. The AID(R112H) mouse model for HIGM provides a powerful and more precise tool than conventional knockout strategies.

Importance of Polη for damage-induced cohesion reveals differential regulation of cohesion establishment at the break site and genome-wide.

Genome integrity depends on correct chromosome segregation, which in turn relies on cohesion between sister chromatids from S phase until anaphase. S phase cohesion, together with DNA double-strand break (DSB) recruitment of cohesin and formation of damage-induced (DI) cohesion, has previously been shown to be required also for efficient postreplicative DSB repair. The budding yeast acetyltransferase Eco1 (Ctf7) is a common essential factor for S phase and DI-cohesion. The fission yeast Eco1 ortholog, Eso1, is expressed as a fusion protein with the translesion synthesis (TLS) polymerase Polη. The involvement of Eso1 in S phase cohesion was attributed to the Eco1 homologous part of the protein and bypass of UV-induced DNA lesions to the Polη part. Here we describe an additional novel function for budding yeast Polη, i.e. formation of postreplicative DI genome-wide cohesion. This is a unique Polη function not shared with other TLS polymerases. However, Polη deficient cells are DSB repair competent, as Polη is not required for cohesion locally at the DSB. This reveals differential regulation of DSB-proximal cohesion and DI genome-wide cohesion, and challenges the importance of the latter for DSB repair. Intriguingly, we found that specific inactivation of DI genome-wide cohesion increases chromosomal mis-segregation at the entrance of the next cell cycle, suggesting that S phase cohesion is not sufficient for correct chromosome segregation in the presence of DNA damage.

Chromosome segregation and double-strand break repair - a complex connection.

Genome stability requires correct chromosome segregation and DNA repair. Failure of these processes leads to cell death or accumulation of chromosomal aberrations, as often observed in tumor cells. An increasing number of observations indicate that segregation and DNA double-strand break (DSB) repair are functionally connected by the Cohesin and Smc5/6 protein complexes. Through their interaction with the duplicated genome, these complexes play essential roles in both chromosome segregation and repair by sister chromatid recombination. Both are also recruited to DSBs, and their chromosomal association is similarly regulated. Interestingly, recent studies of Cohesin suggest that DSB formation could promote proper mitotic chromosome segregation. This is reminiscent of segregation in meiotic cells, which is facilitated by break-induced chromosomal tethering.

Large tundra methane burst during onset of freezing.

Terrestrial wetland emissions are the largest single source of the greenhouse gas methane. Northern high-latitude wetlands contribute significantly to the overall methane emissions from wetlands, but the relative source distribution between tropical and high-latitude wetlands remains uncertain. As a result, not all the observed spatial and seasonal patterns of atmospheric methane concentrations can be satisfactorily explained, particularly for high northern latitudes. For example, a late-autumn shoulder is consistently observed in the seasonal cycles of atmospheric methane at high-latitude sites, but the sources responsible for these increased methane concentrations remain uncertain. Here we report a data set that extends hourly methane flux measurements from a high Arctic setting into the late autumn and early winter, during the onset of soil freezing. We find that emissions fall to a low steady level after the growing season but then increase significantly during the freeze-in period. The integral of emissions during the freeze-in period is approximately equal to the amount of methane emitted during the entire summer season. Three-dimensional atmospheric chemistry and transport model simulations of global atmospheric methane concentrations indicate that the observed early winter emission burst improves the agreement between the simulated seasonal cycle and atmospheric data from latitudes north of 60 degrees N. Our findings suggest that permafrost-associated freeze-in bursts of methane emissions from tundra regions could be an important and so far unrecognized component of the seasonal distribution of methane emissions from high latitudes.

A system for inducible mitochondria-specific protein degradation in vivo.

Targeted protein degradation systems developed for eukaryotes employ cytoplasmic machineries to perform proteolysis. This has prevented mitochondria-specific analysis of proteins that localize to multiple locations, for example, the mitochondria and the nucleus. Here, we present an inducible mitochondria-specific protein degradation system in Saccharomyces cerevisiae based on the Mesoplasma florum Lon (mf-Lon) protease and its corresponding ssrA tag (called PDT). We show that mitochondrially targeted mf-Lon protease efficiently and selectively degrades a PDT-tagged reporter protein localized to the mitochondrial matrix. The degradation can be induced by depleting adenine from the medium, and tuned by altering the promoter strength of the MF-LON gene. We furthermore demonstrate that mf-Lon specifically degrades endogenous, PDT-tagged mitochondrial proteins. Finally, we show that mf-Lon-dependent PDT degradation can also be achieved in human mitochondria. In summary, this system provides an efficient tool to selectively analyze the mitochondrial function of dually localized proteins.

Liquid chromatography/time-of-flight mass spectrometry analysis of postmortem blood samples for targeted toxicological screening.

A liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS) method for targeted toxicological screening in human postmortem blood samples from forensic autopsy cases has been developed, validated and compared with a previously used method using gas chromatography with nitrogen-phosphorus detection (GC-NPD). Separation was achieved within 12 min by high-resolution gradient chromatography. Ions were generated in positive and negative electrospray ionization mode and were detected in 2-GHz single mass spectrometry mode, m/z range 50-1,000. Before injection, 0.25 g blood was prepared by protein precipitation with 500 µL of a mixture of acetonitrile and ethanol containing deuterated internal standards. An in-house database comprising 240 drugs and metabolites was built by analysing solutions from certified standards or other documented reference material available. Identification was based on scoring of retention time, accurate mass measurement and isotopic pattern. Validation was performed on spiked blood samples and authentic postmortem blood samples. The thresholds defined as minimum required performance levels were for most compounds in the range from 0.01 to 0.10 µg/g. Typically, a mass error of less than 2 ppm and a precision of area measurements of less than 5 % coefficient of variation were achieved. Positive identification was confirmed at concentrations up to 500 µg/g. Most compounds were determined in positive ionization mode, but for a limited number of compounds (fewer than 4 %) negative ionization was needed and a few early-eluted compounds could not be identified owing to substantial influence of interferences from the matrix and were thus not included in the screening. A robust and valid toxicological screening by LC-TOF-MS for postmortem blood samples, covering 50 % more compounds, and with higher precision and sensitivity than the previously used screening by GC-NPD was achieved.

Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection.

Homologous recombination enables cells to overcome the threat of DNA double-strand breaks (DSBs), allowing for repair without the loss of genetic information. Central to the homologous recombination repair process is the de novo loading of cohesin around a DSB by its loader complex Scc2/4. Although cohesin's DSB accumulation has been explored in numerous studies, the prerequisites for Scc2/4 recruitment during the repair process are still elusive. To address this question, we combine chromatin immunoprecipitation-qPCR with a site-specific DSB in vivo, in Saccharomyces cerevisiae We find that Scc2 DSB recruitment relies on γH2A and Tel1, but as opposed to cohesin, not on Mec1. We further show that the binding of Scc2, which emanates from the break site, depends on and coincides with DNA end resection. Absence of chromatin remodeling at the DSB affects Scc2 binding and DNA end resection to a comparable degree, further indicating the latter to be a major driver for Scc2 recruitment. Our results shed light on the intricate DSB repair cascade leading to the recruitment of Scc2/4 and subsequent loading of cohesin.

Plasma atropine concentrations associated with decreased intestinal motility in horses.

Introduction: Atropine is an essential part of the treatment protocol for equine uveitis. Topical atropine administration has been associated with decreased intestinal motility and abdominal pain in horses. Experimental studies have indicated that frequent dosing is associated with a higher risk than dosing every 6 h. Unfortunately, no quantitative pharmacodynamic data for inhibition of the equine gut are published. Materials and methods: Eight standardbred horses were assigned to receive either atropine or saline (control) to be infused over 30 min in a two-treatment cross-over design. Atropine concentrations in plasma were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. Intestinal motility was measured using borborygmi frequency and electrointestinography (EIG). Experimental data were analyzed using a non-linear mixed effects model. The model was then used to simulate different dosing regimens. Results: Atropine significantly decreased borborygmi response and EIG response. Six horses developed clinical signs of abdominal pain. The pharmacokinetic typical values were 0.31, 1.38, 0.69, and 1.95 L/kg·h for the volumes of the central, the highly perfused, the scarcely perfused compartments, and the total body clearance, respectively. The pharmacodynamic typical values were 0.31 µg/L and 0.6 and 207 nV27 cpm for the plasma concentration at 50% of the maximum response and the maximum response and the baseline of cecal EIG response, respectively. Six different dosing regimens of topical atropine sulfate to the eye (0.4 and 1 mg every hour, every 3 h, and every 6 h) were simulated. Conclusion: The IV PK/PD data coupled with simulations predict that administration of 1 mg of topical atropine sulfate administered to the eye every hour or every 3 h will lead to atropine accumulation in plasma and decreased intestinal myoelectric activity. Administration every 6 h predicted a safe dosing regimen in full-sized horses. Clinical studies would be valuable to confirm the conclusions. For smaller equids and horses put at risk for colic due to othercauses, droplet bottles that deliver 40 µl of 1% atropine sulfate per drop or less may be used to lower the risk further.

S-phase and DNA damage activated establishment of sister chromatid cohesion--importance for DNA repair.

By holding sister chromatids together from the moment of their formation until their separation at anaphase, the multi subunit protein complex Cohesin guarantees correct chromosome segregation. This S-phase established chromatid cohesion is also essential for repair of DNA double strand breaks (DSB) in postreplicative cells. In addition, Cohesin has to be recruited to a DSB, and new cohesion has to form in response to the damage for repair. When it became clear that cohesion is created de novo in response to DNA breaks, the term "damage induced cohesion" (DI-cohesion) was coined. It is now established that certain factors are needed for establishment of both S-phase and DI-cohesion, while others have been found to be unique for respective process. In addition, post-translational modifications of Cohesin components that are functionally important for cohesion formation, either during S-phase or in response to damage, have recently been identified. Here, we present and discuss the current models for establishment of S-phase and DI-cohesion in the context of their involvement in DSB repair.

The missing pieces for better future predictions in subarctic ecosystems: A Torneträsk case study.

Arctic and subarctic ecosystems are experiencing substantial changes in hydrology, vegetation, permafrost conditions, and carbon cycling, in response to climatic change and other anthropogenic drivers, and these changes are likely to continue over this century. The total magnitude of these changes results from multiple interactions among these drivers. Field measurements can address the overall responses to different changing drivers, but are less capable of quantifying the interactions among them. Currently, a comprehensive assessment of the drivers of ecosystem changes, and the magnitude of their direct and indirect impacts on subarctic ecosystems, is missing. The Torneträsk area, in the Swedish subarctic, has an unrivalled history of environmental observation over 100 years, and is one of the most studied sites in the Arctic. In this study, we summarize and rank the drivers of ecosystem change in the Torneträsk area, and propose research priorities identified, by expert assessment, to improve predictions of ecosystem changes. The research priorities identified include understanding impacts on ecosystems brought on by altered frequency and intensity of winter warming events, evapotranspiration rates, rainfall, duration of snow cover and lake-ice, changed soil moisture, and droughts. This case study can help us understand the ongoing ecosystem changes occurring in the Torneträsk area, and contribute to improve predictions of future ecosystem changes at a larger scale. This understanding will provide the basis for the future mitigation and adaptation plans needed in a changing climate.

Post-translational Regulation of DNA Polymerase η, a Connection to Damage-Induced Cohesion in Saccharomyces cerevisiae.

Double-strand breaks that are induced postreplication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break site and genome-wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome-wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin-dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28 Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo We also demonstrated that Eco1 acetylated polymerase η in vitro Certain nonacetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation, and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as post-translational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.

Impact of a 6-week non-energy-restricted ketogenic diet on physical fitness, body composition and biochemical parameters in healthy adults.

BACKGROUND: The ketogenic diet (KD) is a very low-carbohydrate, high-fat and adequate-protein diet that without limiting calories induces different metabolic adaptations, eg, increased levels of circulating ketone bodies and a shift to lipid metabolism. Our objective was to assess the impact of a 6-week non-energy-restricted KD in healthy adults beyond cohorts of athletes on physical performance, body composition, and blood parameters. METHODS: Our single arm, before-and-after comparison study consisted of a 6-week KD with a previous preparation period including detailed instructions during classes and individual counselling by a dietitian. Compliance with the dietary regimen was monitored by measuring urinary ketones daily, and 7-day food records. All tests were performed after an overnight fast: cardiopulmonary exercise testing via cycle sprioergometry, blood samples, body composition, indirect calorimetry, handgrip strength, and questionnaires addressing complaints and physical sensations. RESULTS: Forty-two subjects aged 37 ± 12 years with a BMI of 23.9 ± 3.1 kg/m2 completed the study. Urinary ketosis was detectable on 97% of the days, revealing very good compliance with the KD. Mean energy intake during the study did not change from the habitual diet and 71.6, 20.9, and 7.7% of total energy intake were from fat, protein, and carbohydrates, respectively. Weight loss was -2.0 ± 1.9 kg (P < 0.001) with equal losses of fat-free and fat mass. VO2peak and peak power decreased from 2.55 ± 0.68 l/min to 2.49 ± 0.69 l/min by 2.4% (P = 0.023) and from 241 ± 57 W to 231 ± 57 W by 4.1% (P < 0.001), respectively, whereas, handgrip strength rose slightly from 40.1 ± 8.8 to 41.0 ± 9.1 kg by 2.5% (P = 0.047). The blood lipids TG and HDL-C remained unchanged, whereas total cholesterol and LDL-C increased significantly by 4.7 and 10.7%, respectively. Glucose, insulin, and IGF-1 dropped significantly by 3.0, 22.2 and 20.2%, respectively. CONCLUSIONS: We detected a mildly negative impact from this 6-week non-energy-restricted KD on physical performance (endurance capacity, peak power and faster exhaustion). Our findings lead us to assume that a KD does not impact physical fitness in a clinically relevant manner that would impair activities of daily living and aerobic training. However, a KD may be a matter of concern in competitive athletes. TRIAL REGISTRATION: DRKS00009605, registered 08 January 2016.

Binding of Ikaros to germline Ig heavy chain gamma1 and epsilon promoters.

Immunoglobulin (Ig) class switching occurs in activated B cells and results in production of antigen-specific IgA, IgE or IgG. It involves a DNA recombination event and is partly regulated by germline (GL) immunoglobulin heavy chain promoters. Ikaros is an abundant nuclear protein expressed in hematopoietic cells. Many different functions have been ascribed to Ikaros, such as transcriptional activation or repression, cell cycle control and tumor suppression. A typical feature of Ikaros is its expression in large clusters in the nucleus of activated lymphocytes. We give evidence that Ikaros can bind to several sites in the germline gamma1 and epsilon immunoglobulin heavy chain promoters, in a cooperative manner. Using a promoter reporter assay, we found evidence that Ikaros can suppress germline gamma1 and epsilon promoter activity in a B cell line. When a mutated non-DNA-binding form of Ikaros was introduced into primary activated B cells by retrovirus transduction, the endogenous Ikaros clusters were disrupted. In spite of this, there was no effect on transcription or Ig class switching. The data are discussed in relation to the different hypotheses for the function of Ikaros.