Role of AID and microRNA-155 in c-myc-IgH Translocations

Chromosome translocations between oncogenes and the immunoglobulin (Ig) region spanning the variable (V), diversity (D) and joining (J) genes (Ig V-JH region) are found in a number of mature B cell lymphomas in humans and mice. The breakpoints are frequently adjacent to the recombination signal sequences (RSSs) targeted by recombinase activating genes 1 and 2 (RAG1/2) during antigen receptor assembly in pre- B cells, suggesting that these translocations might be the result of aberrant V(D)J recombination. However, in mature B cells undergoing AID dependent somatic hypermutation (SHM), duplications or deletions that would necessitate a double strand break make up 6% of all the Ig V-JH region associated somatic mutations. Furthermore, DNA breaks can be detected at this locus in B cells undergoing SHM. To determine whether SHM might induce c-myc to Ig V-JH translocations, we searched for such events in both IL6 transgenic (IL6 tg) and AID-/- IL6 tg mice. Our experiments demonstrate that AID is required for c-myc to Ig V-JH translocations induced by IL6. We also investigated the potential role of microRNAs (miRNAs) in AID mediated processes. MiRNAs are small non-coding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA in vivo we created a knock-in mouse that carries a mutation in the putative microRNA-155 (miR-155) target site in the 3’UTR of AID (AID155 mice). AID155 causes an increase in steady state AID mRNA and protein levels by increasing the half-life of the mRNA resulting in high levels of c-myc-IgH translocations. A similar but more pronounced translocation phenotype was also found in bic/miR-155 /- mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID

Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo

Dendritic cells (DCs) have the capacity to initiate immune responses, but it has been postulated that they may also be involved in inducing peripheral tolerance. To examine the function of DCs in the steady state we devised an antigen delivery system targeting these specialized antigen presenting cells in vivo using a monoclonal antibody to a DC-restricted endocytic receptor, DEC-205. Our experiments show that this route of antigen delivery to DCs is several orders of magnitude more efficient than free peptide in complete Freund\u27s adjuvant (CFA) in inducing T cell activation and cell division. However, T cells activated by antigen delivered to DCs are not polarized to produce T helper type 1 cytokine interferon γ and the activation response is not sustained. Within 7 d the number of antigen-specific T cells is severely reduced, and the residual T cells become unresponsive to systemic challenge with antigen in CFA. Coinjection of the DC-targeted antigen and anti-CD40 agonistic antibody changes the outcome from tolerance to prolonged T cell activation and immunity. We conclude that in the absence of additional stimuli DCs induce transient antigen- specific T cell activation followed by T cell deletion and unresponsiveness

Haploinsufficiency of Activation-Induced Deaminase for Antibody Diversification and Chromosome Translocations both In Vitro and In Vivo

The humoral immune response critically relies on the secondary diversification of antibodies. This diversification takes places through somatic remodelling of the antibody genes by two molecular mechanisms, Class Switch Recombination (CSR) and Somatic Hypermutation (SHM). The enzyme Activation Induced Cytidine Deaminase (AID) initiates both SHM and CSR by deaminating cytosine residues on the DNA of immunoglobulin genes. While crucial for immunity, AID-catalysed deamination is also the triggering event for the generation of lymphomagenic chromosome translocations. To address whether restricting the levels of AID expression in vivo contributes to the regulation of its function, we analysed mice harbouring a single copy of the AID gene (AID+/−). AID+/− mice express roughly 50% of normal AID levels, and display a mild hyperplasia, reminiscent of AID deficient mice and humans. Moreover, we found that AID+/− cells have an impaired competence for CSR and SHM, which indicates that AID gene dose is limiting for its physiologic function. We next evaluated the impact of AID reduction in AID+/− mice on the generation of chromosome translocations. Our results show that the frequency of AID-promoted c-myc/IgH translocations is reduced in AID+/− mice, both in vivo and in vitro. Therefore, AID is haploinsufficient for antibody diversification and chromosome translocations. These findings suggest that limiting the physiologic levels of AID expression can be a regulatory mechanism that ensures an optimal balance between immune proficiency and genome integrity

A role for AID in chromosome translocations between c-myc and the IgH variable region

Chromosome translocations between oncogenes and the region spanning the immunoglobulin (Ig) heavy chain (IgH) variable (V), diversity (D), and joining (J) gene segments (Ig V-JH region) are found in several mature B cell lymphomas in humans and mice. The breakpoints are frequently adjacent to the recombination signal sequences targeted by recombination activating genes 1 and 2 during antigen receptor assembly in pre–B cells, suggesting that these translocations might be the result of aberrant V(D)J recombination. However, in mature B cells undergoing activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM), duplications or deletions that would necessitate a double-strand break make up 6% of all the Ig V-JH region–associated somatic mutations. Furthermore, DNA breaks can be detected at this locus in B cells undergoing SHM. To determine whether SHM might induce c-myc to Ig V-JH translocations, we searched for such events in both interleukin (IL) 6 transgenic (IL-6 tg) and AID−/− IL-6 tg mice. Here, we report that AID is required for c-myc to Ig V-JH translocations induced by IL-6

Exploratory studies of oral and fecal microbiome in healthy human aging.

Growing evidence has linked an altered host fecal microbiome composition with health status, common chronic diseases, and institutionalization in vulnerable older adults. However, fewer studies have described microbiome changes in healthy older adults without major confounding diseases or conditions, and the impact of aging on the microbiome across different body sites remains unknown. Using 16S ribosomal RNA gene sequencing, we reconstructed the composition of oral and fecal microbiomes in young (23-32; mean = 25 years old) and older (69-94; mean = 77 years old) healthy community-dwelling research subjects. In both body sites, we identified changes in minor bacterial operational taxonomic units (OTUs) between young and older subjects. However, the composition of the predominant bacterial species of the healthy older group in both microbiomes was not significantly different from that of the young cohort, which suggests that dominant bacterial species are relatively stable with healthy aging. In addition, the relative abundance of potentially pathogenic genera, such a

KAP-1 promotes resection of broken DNA ends not protected by γ-H2AX and 53BP1 in G1-phase lymphocytes

The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G(1)-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G(1)-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G(1)-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G(1)-phase cells and suggest that there may be species-specific features to this activity

Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing

A large number of miRNAs have recently been discovered in plants and animals. Development of reverse genetic approaches that act to inhibit microRNA function would facilitate the study of this new class of noncoding RNA. Here we show that 2′-O-methyl oligoribonucleotides, but not 2′-deoxyoligonucleotides specifically inactivate the RNAi activity associated with miRNA–protein complexes in human cell extracts as well as in cultured human cells