Using Upd-Seq To Understand Genome-Wide Targeting By The Human Aid/apobec3 Enzymes

AID/APOBEC3 group of cytosine deaminases are central to our innate and adaptive immunity. However, the misregulation of these enzymes may lead to mutations, double-strand breaks, and translocations, that can result in cancer or drug-resistant tumors. Due to the limitations of the current sequencing technologies to detect uracils in the DNA, it is challenging to track the activity of these enzymes. Bhagwat lab has developed a sequencing method called UPD-Seq that can map the genomic uracils. This sequencing method labels and pulls down uracilated fragments of DNA. When the human APOBEC3A or a variant of AID was expressed in E. coli cells and the resulting uracils were mapped, we were able to study the genomic targets of these deaminases and learn about their targeting behavior. Normalized Differential Coverage (NDC) was used in conjunction with other enhancements in the bioinformatics analysis to detect uracil peaks. Uracilation Index as a proxy for mutation proved to be a useful quantitative method to study the deaminase’s genome-wide targeting behavior. A3A and AID both Prefer tRNA coding genes and overwhelmingly target transcription start sites; however, the targeted genes do not have high transcription levels. A3A targets hairpin-forming sequences and this preference can override the enzyme’s nucleotide context preference; AID does not target hairpin-forming sequences. A3A activity is higher at the lagging strand template of replication, while the AID activity does not show any replicative strand bias. We show that A3A Loop1 is important for deaminase activity and substrate specificity. UPD-Seq of an A3A variant with extended loop 1 targets more tRNA coding genes, creates greater replicative strand bias, and prefers different types of hairpin structures compared to the A3A WT. We were also able to use this technology to map the targets and off-targets of the CRISPR/cas9 inspired technology, Cytosine Base Editors (CBE). UPD-Seq is potentially a great tool to test the specificity of the CBEs for when it is used in mammalian systems

A novel class of chemicals that react with abasic sites in DNA and specifically kill B cell cancers

<div><p>Most B cell cancers overexpress the enzyme activation-induced deaminase at high levels and this enzyme converts cytosines in DNA to uracil. The constitutive expression of this enzyme in these cells greatly increases the uracil content of their genomes. We show here that these genomes also contain high levels of abasic sites presumably created during the repair of uracils through base-excision repair. We further show that three alkoxyamines with an alkyne functional group covalently link to abasic sites in DNA and kill immortalized cell lines created from B cell lymphomas, but not other cancers. They also do not kill normal B cells. Treatment of cancer cells with one of these chemicals causes strand breaks, and the sensitivity of the cells to this chemical depends on the ability of the cells to go through the S phase. However, other alkoxyamines that also link to abasic sites- but lack the alkyne functionality- do not kill cells from B cell lymphomas. This shows that the ability of alkoxyamines to covalently link to abasic sites is insufficient for their cytotoxicity and that the alkyne functionality may play a role in it. These chemicals violate the commonly accepted bioorthogonality of alkynes and are attractive prototypes for anti-B cell cancer agents.</p></div

Levels of genomic AP sites and expression genes responsible for the creation and repair of AP sites.

<p>(A) Relative levels of AP sites in the DNA of B-NHL and non-B-NHL cells. The level of AP sites of each cell line is shown relative to the level in HeLa cells (set to 1). **** is P-value <0.0001, and *** is P-value <0.0005. In all cases, the mean and standard deviations are shown. (B) The expression of different genes in Daudi, Raji and HeLa cells relative to the β actin gene set at 100. “*” denotes undetectable expression.</p

Reaction of alkoxyamines with AP sites.

<p>(A) The open-chain aldehyde form of an AP site in DNA reacts with an alkoxyamine (H₂N-O-R). (B) Structures of different alkoxyamines used in this study.</p

Effects of CRT0044876 on APE-1 and Raji cells.

<p>(A) A synthetic oligomer containing a uracil was treated with Ung to create an AP site and the AP site cleaved by APE-1 in the presence or absence of the inhibitor CRT0044876 (100 μM). (B) Raji cells were treated with different concentrations of CRT0044876 dissolved in DMSO and the viability of the cells after 24 hr treatment is presented. “DMSO” represents viability of cells following treatment with 1% DMSO alone.</p

Comparison of the sensitivity of a B-NHL cell line to different alkoxyamines.

<p>(A) Comparison of sensitivity of Daudi cells to MX, ARP or AA3. (B) Reduction in cell growth following treatment of Daudi cells with MX, ARP and AA3. Cells were treated for 24 hr at indicated concentrations of chemicals. Horizontal dotted line represents the starting density of cells. “*” is P-value of <0.05 and “**” is P-value of <0.01. (C) Suppression of AA3 cytotoxicity for Daudi cells by pre-treatment of the cells with MX. (D) Comparison of cytotoxicities of different analogs of AA3 for Daudi cells. In all cases, the viability of untreated cells (NT) is set at 100% and the mean and standard deviation of six replicates are shown (** represents P-value <0.005, n.s. is not significant).</p

Levels of genomic AP sites and expression genes responsible for the creation and repair of AP sites.

<p>(A) Relative levels of AP sites in the DNA of B-NHL and non-B-NHL cells. The level of AP sites of each cell line is shown relative to the level in HeLa cells (set to 1). **** is P-value <0.0001, and *** is P-value <0.0005. In all cases, the mean and standard deviations are shown. (B) The expression of different genes in Daudi, Raji and HeLa cells relative to the β actin gene set at 100. “*” denotes undetectable expression.</p

Insensitivity of G1-arrested Daudi cells to AA3.

<p>Daudi cells grown in normal growth media (-Mimosine), cells blocked in G1 using Mimosine treatment (+Mimosine) and cells released from Mimosine block (Mimosine release) were incubated with AA3 and the cell viability was determined. The numbers were normalized to the viability of cells without AA3 treatment (set to 100). The data are shown as the mean and standard deviation from six replicates (** represents P-value <0.005, n.s. is not significant).</p

Sensitivities of different cells to AA3.

<p>(A) Killing of B-NHL cells by micromolar concentrations of AA3. (B) Killing of B-NHL cells by millimolar concentrations of AA3. (C) Comparison of AA3 sensitivities of Daudi cells, normal human B cells and primary human keratinocytes (HEKn). (D) Comparison of AA3 sensitivity of Daudi cells with non-B cell lines HeLa, MCF-7, MDA-MB 453, A549 and HEK293T. The data in panel A is from triplicates, while the data in the remaining panels is from six replicates. (E) Correlation between AP sites in B-NHL and other cell lines and killing by treatment with 5 mM AA3. These data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185010#pone.0185010.g002" target="_blank">Fig 2</a>, and parts B and D of this figure. In all cases, the mean and standard deviations are shown.</p

Detection of γ-H2AX in Daudi cells following AA3 treatment.

<p>(A) Daudi cells were stained with anti-γ-H2AX antibodies and visualized using immunofluorescence. Untreated cells are compared with AA3- or AA6-treated, or Phleomycin-treated cells. Representative images of cells stained with DAPI (blue), anti-γ-H2AX antibodies labeled with Cy3 (red) and the overlay of the two images are shown. (B) Quantification of γ-H2AX nuclear fluorescence intensity. The total number of cells used for quantification- Untreated, 74; AA6-treated, 70; AA3-treated, 184 and Phleomycin-treated, 122. Mean intensity and standard deviation for each cell type is shown.</p