31 research outputs found
The Interaction between AID and CIB1 Is Nonessential for Antibody Gene Diversification by Gene Conversion or Class Switch Recombination
Activation-induced deaminase (AID) initiates somatic hypermutation, gene conversion and class switch recombination by deaminating variable and switch region DNA cytidines to uridines. AID is predominantly cytoplasmic and must enter the nuclear compartment to initiate these distinct antibody gene diversification reactions. Nuclear AID is relatively short-lived, as it is efficiently exported by a CRM1-dependent mechanism and it is susceptible to proteasome-dependent degradation. To help shed light on mechanisms of post-translational regulation, a yeast-based screen was performed to identify AID-interacting proteins. The calcium and integrin binding protein CIB1 was identified by sequencing and the interaction was confirmed by immunoprecipitation experiments. The AID/CIB1 resisted DNase and RNase treatment, and it is therefore unlikely to be mediated by nucleic acid. The requirement for CIB1 in AID-mediated antibody gene diversification reactions was assessed in CIB1-deficient DT40 cells and in knockout mice, but immunoglobulin gene conversion and class switch recombination appeared normal. The DT40 system was also used to show that CIB1 over-expression has no effect on gene conversion and that AID-EGFP subcellular localization is normal. These combined data demonstrate that CIB1 is not required for AID to mediate antibody gene diversification processes. It remains possible that CIB1 has an alternative, a redundant or a subtle non-limiting regulatory role in AID biology
AID can restrict L1 retrotransposition suggesting a dual role in innate and adaptive immunity
Retrotransposons make up over 40% of the mammalian genome. Some copies are still capable of mobilizing and new insertions promote genetic variation. Several members of the APOBEC3 family of DNA cytosine deaminases function to limit the replication of a variety of retroelements, such as the long-terminal repeat (LTR)-containing MusD and Ty1 elements, and that of the non-LTR retrotransposons, L1 and Alu. However, the APOBEC3 genes are limited to mammalian lineages, whereas retrotransposons are far more widespread. This raises the question of what cellular factors control retroelement transposition in species that lack APOBEC3 genes. A strong phylogenetic case can be made that an ancestral activation-induced deaminase (AID)-like gene duplicated and diverged to root the APOBEC3 lineage in mammals. Therefore, we tested the hypothesis that present-day AID proteins possess anti-retroelement activity. We found that AID can inhibit the retrotransposition of L1 through a DNA deamination-independent mechanism. This mechanism may manifest in the cytoplasmic compartment co- or posttranslationally. Together with evidence for AID expression in the ovary, our data combined to suggest that AID has innate immune functions in addition to its integral roles in creating antibody diversity
AID interacts with the calcium and integrin binding protein 1.
<p><b>A</b>. myc-CIB1 co-IPs AID-EGFP but not EGFP only (left and right lanes, respectively). A myc-specific monoclonal antibody was used to precipitate complexes, and AID-GFP was detected with an α-GFP polyclonal antibody. <b>B</b>. AID-EGFP co-IPs myc-CIB1 in a DNase I- and RNase A-resistant manner. An α-GFP monoclonal antibody was used to precipitate AID-GFP, and myc-CIB1 was detected with an α-myc monoclonal antibody. <b>C</b>. AID-STZ pulls down endogenous CIB1 from HEK-293T cell extracts. IgG Sepharose was used to pull-down STZ complexes, and CIB1 was detected using an α-CIB1 polyclonal antibody. AID-STZ and GFP-STZ were detected with an α-strep antibody. A low level of non-specific background was observed in the vicinity of AID-STZ. For cell lysate (input) control blots, two panels are shown because GFP-STZ is expressed over 100-fold better than AID-STZ. A quantification of the input versus pull-down signal indicated that 1% of cellular CIB1 can be pulled-down with AID complexes when IgG sepharose beads are limiting.</p
CIB1 over-expression in DT40.
<p><b>A</b>. An IGC fluctuation analysis showing the percentage of surface Ig-positive cells in subclone cultures over-expressing human (h) or chicken (c) CIB1. Each X represents data from an individual subclone and the labeled horizontal bars report the medians for each data set. <b>B</b>. CIB1 over-expression confirmed by immunoblotting. Loading was controlled by stripping and re-probing the blot with an α-tubulin antibody.</p
AID localization in CIB<sup>−/−</sup> DT40 cells.
<p><b>A</b>. AID-EGFP localization in CIB1<sup>+/+</sup> DT40. <b>B</b>. AID-EGFP localization in CIB1<sup>−/−</sup> DT40. Images were taken using a 40× objective and the scale bars indicate 10 µm.</p
CIB1 is dispensable for immunoglobulin gene conversion in DT40.
<p><b>A</b>. Schematic showing the constructs used to replace exons 5 and 6 of <i>CIB1</i> with the indicated drug resistance cassettes. The positions of the allele-specific PCR primers for genotyping and the XhoI sites and the position of the 3′ external probe used for Southern blot analysis are shown. <b>B</b>. An agarose gel image showing the allele-specific PCR products from CIB1+/+, +/−, and −/− cell lines. <b>C</b>. An agarose gel image of CIB1-specific RT-PCR products from CIB1+/+, +/−, and −/− cell lines. AID-specific reactions were used to demonstrate the integrity of each cDNA preparation. <b>D</b>. An IGC fluctuation analysis showing the percentage of surface Ig-positive cells in subclone cultures of the indicated genotype. Each X represents data from an individual subclone and the labeled horizontal bars report the medians for each data set.</p
CIB1 is dispensable for CSR in mice.
<p><b>A</b>. Relative levels of each antibody isotype in sera from CIB1<sup>+/+</sup> or CIB1<sup>−/−</sup> mice as measured by ELISA. The CIB1<sup>−/−</sup> data were normalized to the mean antibody levels in sera from wildtype (WT) littermates (arbitrarily set to 1 for comparison). Each X represents data from an independent animal and the horizontal bars and labels report the median values (n = 3 for CIB<sup>+/+</sup> and n = 6 for CIB<sup>−/−</sup>). <b>B</b>. IgM to IgG1 CSR <i>ex vivo</i>. B-cells were isolated from the spleens of CIB1<sup>+/+</sup> or CIB1<sup>−/−</sup> mice, cultured for 4 days in the presence of LPS and IL-4, and analyzed by α-IgG1-PE labeling and flow cytometry. Each X represents data from an independent animal and the horizontal bars and labels report the median values. <b>C</b>. Images of hematoxylin and eosin stained sections of spleen isolated from CIB1<sup>+/+</sup> and CIB1<sup>−/−</sup> mice. Scale bars indicate 500 µm.</p
TAL Effector Specificity for base 0 of the DNA Target Is Altered in a Complex, Effector- and Assay-Dependent Manner by Substitutions for the Tryptophan in Cryptic Repeat –1
<div><p>TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of <i>Ralstonia solanacearum</i>, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5’ base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.</p> </div
Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA
<div><p>Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the <i>Nicotiana benthamiana</i> ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were <10bp; in contrast, 88% of insertions generated through DNA delivery were >10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from <i>Arabidopsis thaliana</i> genes to the TALEN coding sequence. UTRs from an <i>A</i>. <i>thaliana</i> adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing.</p></div
Alignment of N-terminal portions of multiple TAL effector crystal structures shows a conserved conformation for W232 consistent with an important interaction with the 0<sup>th</sup> position T.
<p>The N terminus of TAL effector PthXo1 bound to its DNA target (PDB structure 3UGM) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082120#B4" target="_blank">4</a>] is shown in blue, the N terminus of unbound artificial TAL effector dTALE2 (PDB structure 4HPZ) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082120#B26" target="_blank">26</a>] in red, and the N terminus of artificial TAL effector dHAX3 bound to a DNA-RNA hybrid (PDB structure 4GG4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082120#B5" target="_blank">5</a>] in brown. DNA is from structure 3UGM. Side chains are shown for W232 as well as arginines at positions 236 and 266, which make non-specific contacts to the nucleic acid backbone. T<sub>0</sub>, the 0<sup>th</sup> position thymine. G<sub>-1</sub>, a guanine 5’ of T<sub>0</sub>. </p