Localized DNA Demethylation at Recombination Intermediates during Immunoglobulin Heavy Chain Gene Assembly

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (DH) and joining (JH) gene segments were methylated, DJH junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eμ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination

Analysis of Mice Lacking DNaseI Hypersensitive Sites at the 5′ End of the IgH Locus

The 5′ end of the IgH locus contains a cluster of DNaseI hypersensitive sites, one of which (HS1) was shown to be pro-B cell specific and to contain binding sites for the transcription factors PU.1, E2A, and Pax5. These data as well as the location of the hypersensitive sites at the 5′ border of the IgH locus suggested a possible regulatory function for these elements with respect to the IgH locus. To test this notion, we generated mice carrying targeted deletions of either the pro-B cell specific site HS1 or the whole cluster of DNaseI hypersensitive sites. Lymphocytes carrying these deletions appear to undergo normal development, and mutant B cells do not exhibit any obvious defects in V(D)J recombination, allelic exclusion, or class switch recombination. We conclude that deletion of these DNaseI hypersensitive sites does not have an obvious impact on the IgH locus or B cell development

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation

A tissue-specific transcriptional enhancer, Eμ, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Eμ results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Eμ-independent and Eμ-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression

Preliminary Results of Terabit-per-second Long-Range Free-Space Optical Transmission Experiment THRUST

Future Very High Throughput Satellite Systems (VHTS) will perform at several Tbit/s throughput and thus face the challenge of limited feeder-link spectrum. Whereas with conventional RF feeder links several tens of ground gateway stations would be required, the total capacity can alternatively be linked through a single optical ground station using Dense Wavelength Division Multiplexing (DWDM) techniques as known from terrestrial fiber communications. While intermittent link blockage by clouds can be compensated by ground station diversity, the optical uplink signal is directly affected by scintillation and beam wander induced by the atmospheric index-of-refraction turbulence. The transmission system must be capable to mitigate these distortions by according high-speed tracking and fading compensation techniques. We report on the design of a near-ground long-range (10km) atmospheric transmission test-bed which is, with its relatively low elevation of 1.8 degrees, exemplary for a worst case GEO uplink scenario. The transmitting side of the test-bed consists of a single telescope with a a fine pointing assembly in order to track the atmospheric angle-of-arrival and precisely aim towards the beacon of the receiver. On the other side of the test-bed, the receiver telescope is also capable of fine pointing by tracking the transmitted signal. The GEO uplink scenario is modelled by a precise scaling of the beam divergence and the receiver’s field of view as well as by the beacon offset to model the point-ahead angle. In order to make the experimental test-bed correspond to an actual feeder link scenario, the link budget as well as the turbulence profile of the experimental scenario are modelled and compared to the GEO uplink. Several DWDM channels are multiplexed to reach the total link capacity of above one Tbit/s. We report on the preparatory lab tests, and the free-space test-bed design and verification, of the Terabit Throughput Satellite Technology Project THRUST

Optical GEO Feeder Link Design

Abstract: Telecommunication satellites must follow the advances of terrestrial network capacities and increase their total throughputs in order to remain competitive. This paper shows how the terrestrial fibre technology at 1550-nm wavelength can be extended to support an optical feeder link between ground and a GEO satellite. With 100 Gb/s in a single direction, an optical uplink would outperform the near-term Ka-band systems. Mitigation techniques against clouds and turbulence are described. The impact of satellite transparency on the optical transmission system is discussed. To increase the profitability of the optical solution, cost and link availability associated with various ground station networks should be carefully assessed

S-S synapsis during class switch recombination is promoted by distantly located transcriptional elements and activation-induced deaminase.

International audienceMolecular mechanisms underlying synapsis of activation-induced deaminase (AID)-targeted S regions during class switch recombination (CSR) are poorly understood. By using chromosome conformation capture techniques, we found that in B cells, the Emicro and 3'Ealpha enhancers were in close spatial proximity, forming a unique chromosomal loop configuration. B cell activation led to recruitment of the germline transcript (GLT) promoters to the Emicro:3'Ealpha complex in a cytokine-dependent fashion. This structure facilitated S-S synapsis because Smicro was proximal to Emicro and a downstream S region was corecruited with the targeted GLT promoter to Emicro:3'Ealpha. We propose that GLT promoter association with the Emicro:3'Ealpha complex creates an architectural scaffolding that promotes S-S synapsis during CSR and that these interactions are stabilized by AID. Thus, the S-S synaptosome is formed as a result of the self-organizing transcription system that regulates GLT expression and may serve to guard against spurious chromosomal translocations

The RNA helicase DDX3X is an essential mediator of innate antimicrobial immunity.

DExD/H box RNA helicases, such as the RIG-I-like receptors (RLR), are important components of the innate immune system. Here we demonstrate a pivotal and sex-specific role for the heterosomal isoforms of the DEAD box RNA helicase DDX3 in the immune system. Mice lacking DDX3X during hematopoiesis showed an altered leukocyte composition in bone marrow and spleen and a striking inability to combat infection with Listeria monocytogenes. Alterations in innate immune responses resulted from decreased effector cell availability and function as well as a sex-dependent impairment of cytokine synthesis. Thus, our data provide further in vivo evidence for an essential contribution of a non-RLR DExD/H RNA helicase to innate immunity and suggest it may contribute to sex-related differences in resistance to microbes and resilience to inflammatory disease

DNA methylation status of DNase 1 hypersensitive sites 5′ of DFL16.1.

<p>Three newly identified DNase 1 hypersensitive sites are located at approximately 6–6.5, 4–4.5, and 0.4–1.3 kb 5′ of DFL16.1. Genomic DNA from primary RAG-deficient pro-B cells and DP (CD4⁺ CD8⁺) thymocytes were used in bisulfite mapping experiments to examine CpG methylation. Five amplicons covering regions between 3 kb and 7 kb 5′ of DFL16.1 were modified, cloned, and sequenced. The distribution of CpG dinucleotides within each amplicon are noted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475.s003" target="_blank">Figure S3</a>. Filled and open circles represent methylated and unmethylated residues, respectively. Pie charts summarize the percentage of methylated alleles at each position.</p

DNA methylation of IgH alleles in thymocytes.

<p>CD4⁺CD8⁺ thymocytes from C57BL/6 mice were enriched by adsorption to PNA-coated plastic plates. Genomic DNA purified from these cells was used in bisulfite modification analysis. Amplicons corresponding to unrearranged parts of IgH allele (A) and DJ_H junctions (B) were cloned and sequenced. Circles and squares depict CpGs corresponding to D_H and J_H1 cytosines, respectively. Filled circles/squares correspond to methylated cytosines; pie charts summarize the percentage of methylated cytosines at each position except where the number of sequenced alleles falls below 12, indicated by asterisks. Untemplated CpGs incorporated during VDJ recombination were found to be methylated (filled diamonds). Data shown were obtained from two independent preparations of thymocytes, using one mouse per experiment.</p