Nasim A. Begum

Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1

Activation-induced cytidine deaminase (AID) is a molecule central to initiating class switch recombination, somatic hypermutation, and gene conversion of Ig genes. However, its mechanism to initiate these genetic alterations is still unclear. AID can convert cytosine to uracil on either mRNA or DNA and is involved in DNA cleavage. Although these events are expected to take place in the nucleus, overexpressed AID was found predominantly in the cytoplasm. Here, we demonstrated that AID is a nucleocytoplasmic shuttling protein with a bipartite nuclear localization signal and a nuclear export signal in its N and C termini, respectively. In addition to previously identified genetic, structural, and biochemical similarities of AID with apolipoprotein B mRNA editing catalytic polypeptide 1, an RNA editing enzyme of ApoB100 mRNA, the present finding provides another aspect to their resemblance, suggesting that both may have homologous reaction mechanisms.

Separate domains of AID are required for somatic hypermutation and class-switch recombination

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM). Mutants with changes in the C-terminal region of AID retain SHM but lose CSR activity. Here we describe five mutants with alterations in the N-terminal region of AID that caused selective deficiency in SHM but retained CSR, suggesting that the CSR and SHM activities of AID may dissociate via interaction of CSR- or SHM-specific cofactors with different domains of AID. Unlike cells expressing C-terminal AID mutants, B cells expressing N-terminal AID mutants had mutations in the switch μ region, indicating that such mutations are generated by reactions involved in CSR but not SHM. Thus, we propose that separate domains of AID interact with specific cofactors to regulate these two distinct genetic events in a target-specific way.

Mycoplasma fermentans Lipoprotein M161Ag-Induced Cell Activation Is Mediated by Toll-Like Receptor 2: Role of N-Terminal Hydrophobic Portion in its Multiple Functions

M161Ag is a 43-kDa surface lipoprotein of Mycoplasma fermentans, serving as a potent cytokine inducer for monocytes/macrophages, maturing dendritic cells (DCs), and activating host complement on affected cells. It possesses a unique N-terminal lipo-amino acid, S-diacylglyceryl cysteine. The 2-kDa macrophage-activating lipopeptide-2 (MALP-2), recently identified as a ligand for Toll-like receptor 2 (TLR2), is derived from M161Ag. In this study, we identified structural motifs sustaining the functions of M161Ag using wild-type and unlipidated rM161Ag with (SP+) or without signal peptides (SP−). Because the SP+ rM161Ag formed dimers via 25Cys, we obtained a monomeric form by mutagenesis (SP+C25S). Only wild type accelerated maturation of human DCs as determined by the CD83/86 criteria, suggesting the importance of the N-terminal fatty acids for this function. Wild-type and the SP+ form of monomer induced secretion of TNF-α and IL-12 p40 by human monocytes and DCs. Either lipid or signal peptide at the N-terminal portion of monomer was required for expression of this function. In contrast, murine macrophages produced TNF-α in response to wild type, but not to any recombinant form of M161Ag, suggesting the species-dependent response to rM161Ag. Wild-type and both monomeric and dimeric SP+ forms possessed the ability to activate complement via the alternative pathway. Again, the hydrophobic portion was associated with this function. These results, together with the finding that macrophages from TLR2-deficient mice did not produce TNF-α in response to M161Ag, infer that the N-terminal hydrophobic structure of M161Ag is important for TLR2-mediated cell activation and complement activation.

Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus.

Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-β production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By gene-silencing analysis, DI RNA activated the RIG-I/MDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-β mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-β mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells.

Discovery of activation-induced cytidine deaminase, the engraver of antibody memory.

Discovery of activation-induced cytidine deaminase (AID) paved a new path to unite two genetic alterations induced by antigen stimulation; class switch recombination (CSR) and somatic hypermutation (SHM). AID is now established to cleave specific target DNA and to serve as engraver of these genetic alterations. AID of a 198-residue protein has four important domains: nuclear localization signal and SHM-specific region at the N-terminus; the alpha-helical segment (residue 47-54) responsible for dimerization; catalytic domain (residues 56-94) shared by all the other cytidine deaminase family members; and nuclear export signal overlapping with class switch-specific domain at the C-terminus. Two alternative models have been proposed for the mode of AID action; whether AID directly attacks DNA or indirectly through RNA editing. Lines of evidence supporting RNA editing hypothesis include homology in various aspects with APOBEC1, a bona fide RNA editing enzyme as well as requirement of de novo protein synthesis for DNA cleavage by AID in CSR and SHM. This chapter critically evaluates DNA deamination hypothesis and describes evidence to indicate UNG is involved not in DNA cleavage but in DNA repair of CSR. In addition, UNG appears to have a noncanonical function through interaction with an HIV Vpr-like protein at the WXXF motif. Taken together, RNA editing hypothesis is gaining the ground.

B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers

Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes, among other genes. We investigated the transcriptional regulation of Aicda (which encodes AID) in class switch–inducible CH12F3-2 cells and found that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5′ promoter region. The 5′ upstream region contained functional motifs for the response to signaling by cytokines, the ligand for the costimulatory molecule CD40 or stimuli that activated the transcription factor NF-κB. The first intron contained functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell–specific activator Pax5 and E-box-binding proteins. Our results show that Aicda is regulated by the balance between B cell–specific and stimulation-responsive elements and ubiquitous silencers.

Mycobacterium bovis BCG Cell Wall-Specific Differentially Expressed Genes Identified by Differential Display and cDNA Subtraction in Human Macrophages

ABSTRACT We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.

Histone3 lysine4 trimethylation regulated by the facilitates chromatin transcription complex is critical for DNA cleavage in class switch recombination

Ig class switch recombination (CSR) requires expression of activation-induced cytidine deaminase (AID) and transcription through target switch (S) regions. Here we show that knockdown of the histone chaperone facilitates chromatin transcription (FACT) completely inhibited S region cleavage and CSR in IgA-switch-inducible CH12F3-2A B cells. FACT knockdown did not reduce AID or S region transcripts but did decrease histone3 lysine4 trimethylation (H3K4me3) at both the Sμ and Sα regions. Because knockdown of FACT or H3K4 methyltransferase cofactors inhibited DNA cleavage in H3K4me3-depleted S regions, H3K4me3 may serve as a mark for recruiting CSR recombinase. These findings revealed an unexpected evolutionary conservation between CSR and meiotic recombination.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5′ to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Sμ region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes

Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.