Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues: implications for epigenetic reprogramming

DNA deaminases of the Aid/Apobec family convert cytosine into uracil and play key roles in acquired and innate immunity. The epigenetic modification by methylation of cytosine in CpG dinucleotides is also mutagenic, but this is thought to occur by spontaneous deamination. Here we show that Aid and Apobec1 are 5-methylcytosine deaminases resulting in a thymine base opposite a guanine. Their action can thus lead to C --> T transition mutations in methylated DNA, or in conjunction with repair of the T:G mismatch, to demethylation. The Aid and Apobec1 genes are located in a cluster of pluripotency genes including Nanog and Stella and are co-expressed with these genes in oocytes, embryonic germ cells, and embryonic stem cells. These results suggest that Aid and perhaps some of its family members may have roles in epigenetic reprogramming and cell plasticity. Transition in CpG dinucleotides is the most frequent mutation in human genetic diseases, and sequence context analysis of CpG transitions in the APC tumor suppressor gene suggests that DNA deaminases may play a significant role in tumor etiology.     

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.     

Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5

Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.     

A chimeric satellite transgene sequence is inefficiently targeted by viroid-induced DNA methylation in tobacco

In plants, transgenes containing Potato spindle tuber viroid (PSTVd) cDNA sequences were efficient targets of PSTVd infection-mediated RNA-directed DNA methylation. Here, we demonstrate that in PSTVd-infected tobacco plants, a 134 bp PSTVd fragment (PSTVd-134) did not become densely methylated when it was inserted into a chimeric Satellite tobacco mosaic virus (STMV) construct. Only about 4–5% of all cytosines (Cs) of the PSTVd-134 were methylated when flanked by satellite sequences. In the same plants, C methylation was approximately 92% when the PSTVd-134 was in a PSTVd full length sequence context and roughly 33% when flanked at its 3′ end by a 19 bp PSTVd and at its 5′ end by a short viroid-unrelated sequence. In addition, PSTVd small interfering RNAs (siRNAs) produced from the replicating viroid failed to target PSTVd-134-containing chimeric STMV RNA for degradation. Satellite RNAs appear to have adopted secondary structures that protect them against RNA interference (RNAi)—mediated degradation. Protection can be extended to short non-satellite sequences residing in satellite RNAs, rendering them poor targets for nuclear and cytoplasmic RNAi induced in trans.     

A specific viral DNA sequence is stably integrated in herpesvirus oncogenically transformed cells

The integration patterns of viral DNA sequences in three hamster embryo cell lines independently derived by transformation with equine herpesvirus type 1 (EHV-1) have been investigated by DNA blot hybridization analyses for the restriction enzymes Eco RI, Bgl II, Xba I and Bam HI with ³²P-labeled selected DNAs from a collection of cloned EHV-1 restriction enzyme fragments as probes. These EHV-1-transformed cell lines contained subgenomic portions of the viral genome in an integrated state at multiple sites in the host genome. At least one copy of a viral DNA sequence mapping colinearly from 0.32 to 0.38 map units within the EHV-1 genome was common among these three EHV-1 transformed cell lines. The 0.32–0.38 viral DNA sequence was maintained stably even after 125 cell passages, whereas sequences from other positions in the EHV-1 genome were lost progressively during continued cell passage. The significance of the findings that these oncogenically transformed cell lines harbor a specific region of the EHV-1 genome is discussed with regard to stable maintenance of the oncogenically transformed state.     

Muscle FBPase is targeted to nucleus by its 203KKKGK207 sequence

It has been recently found that muscle fructose 1,6‐bisphosphatase (FBPase) is actively transported into cells' nuclei. Results of an analysis in silico of muscle FBPase structure gave rise to a hypothesis that sequence ₂₀₃KKKGK₂₀₇ is responsible for nuclear targeting of the enzyme. To test this, HL‐1 cardiomyocytes were transfected with FITC‐labeled muscle FBPase constructs, bearing mutations within the putative nuclear localization signal (NLS). Results revealed that integrity of the ₂₀₃KKKGK₂₀₇ motif is critical to nuclear targeting of muscle FBPase and even a single amino‐acid change within this sequence results in significant decrease of nuclear accumulation of the enzyme. Although it has long been recognized as a canonical NLS in theoretical and computational research, to the best of our knowledge this is the first experimental evidence confirming that the KKKGK motif can act as a functional NLS in a protein. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine

Activation-induced deaminase (AID) is required for both immunoglobulin class switch recombination and somatic hypermutation. AID is known to deaminate cytidines in single-stranded DNA, but the relationship of this step to the class switch or somatic hypermutation processes is not entirely clear. We have studied the activity of a recombinant form of the mouse AID protein that was purified from a baculovirus expression system. We find that the length of the single-stranded DNA target is critical to the action of AID at the Cs positioned anywhere along the length of the DNA. The DNA sequence surrounding a given C influences AID deamination efficiency. AID preferentially deaminates Cs in the WRC motif, and additionally has a small but consistent preference for purine at the position after the WRC, thereby favoring WRCr (the lowercase r corresponds to the smaller impact on activity).     

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction

Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.     

How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

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Activation-induced cell death in T cell hybridomas is due to apoptosis. Morphologic aspects and DNA fragmentation.

Some T cell hybridomas, upon activation via the TCR, rapidly undergo cell death. In this paper, we demonstrate that this activation-induced cell death (AICD) is accompanied by morphologic changes seen at the electron and light microscopy levels. The most striking changes are an extensive condensation of the chromatin and formation of membrane blebs. In addition to the morphologic changes, a significant portion of genomic DNA is broken at an interval of approximately 200 bp, producing a ladder of oligonucleosome-sized fragments after gel electrophoresis. Taken together, these observations indicate that AICD proceeds via apoptosis, or programmed cell death. This is additionally supported by the observation that AICD-associated phenomena are at least partially inhibited by cycloheximide or actinomycin D. Curiously, AICD and its associated DNA fragmentation are completely inhibited by aurintricarboxylic acid, a known nuclease inhibitor. The possible relationship between AICD in vitro, and the negative selection process (wherein selection may proceed via AICD of developing, autoreactive thymocytes) is discussed.     

Activity of FEN1 endonuclease on nucleosome substrates is dependent upon DNA sequence but not flap orientation

We demonstrated previously that human FEN1 endonuclease, an enzyme involved in excising single-stranded DNA flaps that arise during Okazaki fragment processing and base excision repair, cleaves model flap substrates assembled into nucleosomes. Here we explore the effect of flap orientation with respect to the surface of the histone octamer on nucleosome structure and FEN1 activity in vitro. We find that orienting the flap substrate toward the histone octamer does not significantly alter the rotational orientation of two different nucleosome positioning sequences on the surface of the histone octamer but does cause minor perturbation of nucleosome structure. Surprisingly, flaps oriented toward the nucleosome surface are accessible to FEN1 cleavage in nucleosomes containing the Xenopus 5S positioning sequence. In contrast, neither flaps oriented toward nor away from the nucleosome surface are cleaved by the enzyme in nucleosomes containing the high-affinity 601 nucleosome positioning sequence. The data are consistent with a model in which sequence-dependent motility of DNA on the nucleosome is a major determinant of FEN1 activity. The implications of these findings for the activity of FEN1 in vivo are discussed.     

Activation-induced cytidine deaminase-dependent DNA breaks in class switch recombination occur during G1 phase of the cell cycle and depend upon mismatch repair

Ab class switching occurs by an intrachromosomal recombination and requires generation of double-strand breaks (DSBs) in Ig switch (S) regions. Activation-induced cytidine deaminase (AID) converts cytosines in S regions to uracils, which are excised by uracil DNA glycosylase (UNG). Repair of the resulting abasic sites would yield single-strand breaks (SSBs), but how these SSBs are converted to DSBs is unclear. In mouse splenic B cells, we find that AID-dependent DSBs occur in Smu mainly in the G(1) phase of the cell cycle, indicating they are not created by replication across SSBs. Also, G(1) phase cells express AID, UNG, and mismatch repair (MMR) proteins and possess UNG activity. We find fewer S region DSBs in MMR-deficient B cells than in wild-type B cells, and still fewer in MMR-deficient/SmuTR(-/-) B cells, where targets for AID are sparse. These DSBs occur predominantly at AID targets. We also show that nucleotide excision repair does not contribute to class switching. Our data support the hypothesis that MMR is required to convert SSBs into DSBs when SSBs on opposite strands are too distal to form DSBs spontaneously.     

Activation-induced cytidine deaminase expression in CD4+ T cells is associated with a unique IL-10-producing subset that increases with age

Activation-induced cytidine deaminase (AID), produced by the Aicda gene, is essential for the immunoglobulin gene (Ig) alterations that form immune memory. Using a Cre-mediated genetic system, we unexpectedly found CD4(+) T cells that had expressed Aicda (exAID cells) as well as B cells. ExAID cells increased with age, reaching up to 25% of the CD4(+) and B220(+) cell populations. ExAID B cells remained IgM(+), suggesting that class-switched memory B cells do not accumulate in the spleen. In T cells, AID was expressed in a subset that produced IFN-γ and IL-10 but little IL-4 or IL-17, and showed no evidence of genetic mutation. Interestingly, the endogenous Aicda expression in T cells was enhanced in the absence of B cells, indicating that the process is independent from the germinal center reaction. These results suggest that in addition to its roles in B cells, AID may have previously unappreciated roles in T-cell function or tumorigenesis.