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. 2019 Nov 1:10:2540.
doi: 10.3389/fimmu.2019.02540. eCollection 2019.

Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation

Ryan Costello  1 , Jose F Cantillo  1 , Amy L Kenter  1
Affiliations

Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation

Ryan Costello et al. Front Immunol. .

Abstract

Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.

Keywords: B cells; CRISPR; DT40; Ig; somatic hypermutation; uracil DNA glycosylase.

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Figures

Figure 1
Figure 1
Evolutionary conservation of MBD4 glycosylase domain. (A) Schematic (drawn to scale) of MBD4 protein in human (Homo sapiens), chicken (Gallus gallus), mouse (Mus musculus), ghost shark (Callorhinchus milii), platypus (Ornithorhynchus anatinus), ocean coelacanth (Latimeria chalumnae), and green aphid (Schizaphis graminum). The methyl binding domain (MBD) (black box), MLH1 binding domain (blue oval) and glycosylase domain (red box) are indicated as appropriate and amino acid (aa) number is listed. (B) The annotated human glycosylase domain exons 4–8 (aa 401–580) was used as the original sequence. Multiple aa alignments were performed showing conservation with chicken aa 237-416 (94.4%), mouse aa 375–554 (93.3%), ghost shark aa 347–528 (87.2%), platypus aa 1–108 (92.6%), ocean coelacanth aa 259–438 (90.6%) and green aphid aa 59–219 (70.4%). Conserved aa are indicated by dashes and non-conserved aa are noted using the single letter aa code. The MLH1 binding domain is boxed and the predicted catalytic amino acids in the glycosylase domain are numbered R368 (1), Y540 (2), D560 (3) and K562 (4) using the human MBD4 aa numbering system. (C) (Left panel) The three-dimensional (3D) crystal structure of MBD4 glycosylase domain from human (green) (27) and the predicted chicken (dark salmon) structure were aligned using PyMOL with catalytic amino acids shown in red (human) and blue (chicken). (Right panel) Comparison of the crystal structure involving the catalytic aa, R368 (1), Y540 (2), D560 (3), and K562 (4) in the human (red) and chicken (blue) glycosylase domains is shown in high magnification.
Figure 2
Figure 2
CRISPR/Cas9 genome generated deletions in the chicken MBD4 glycosylase domain of DT40 cells. (A) Schematic of human and chicken MBD4 exons (not drawn to scale) Exon numbers are given in boxes with amino acid (aa)s in each exon labeled. The glycosylase domain is highlighted in red and guide (g) RNA in chicken exon 5 annotated. (B) Schematic of Mbd4 gene showing exon 5 targeted by CRISPR/Cas9 editing (not drawn to scale). Mbd4 PCR amplification using primers F1 and R1 was used to identify indels. The gRNA sequence is underlined along the parental DNA (upper) strand and catalytic aa Y376 (lower strand) shown in blue. HhH motif is displayed by solid black line. Deletions in the Mbd4 gene for clones Mbd4Δ/Δ.14 (12 bp deletion), Mbd4Δ/Δ.11 (17 bp deletion), and Mbd4Δ/Δ.12 (62 bp deletion), as compared to the parental sequence (aa 357–379) and predicted aa sequences are shown. (C) The predicted 3D structure of the chicken MBD4 glycosylase domain (aa262-416) is compared to mutated clones, as indicated. Critical amino acids in WT MBD4 are numbered K304 (1), Y376 (2), D396 (3), and K398 (4). Amino acids G372-K375 are present only in WT.
Figure 3
Figure 3
SHM is elevated in DT40 derived MBD4Δ/Δ clones. (A) Schematic of exons 4, 5, and 6 in the Mbd4 gene and primers F2 and R2 used for RT-PCR analysis. gRNA sequence is indicated by a line (g) in early exon 5 (upper panel). Semi-quantitative RT-PCR analysis of Mbd4 exons 5–6 in parental and deleted clones (middle panel). QRT-PCR analysis of 18S RNA was used as a loading control (lower panel). 18S sample values were normalized to parental values which were set to 1. Samples were assayed in triplicate and were averaged, and SEMs are shown. (B) Cell proliferation assays. Cells (1 × 105/ml) were grown in culture for 96 h and live cells were counted in triplicate every 24 h. The data shown is the average and is representative of 3 independent samples. (C) IgM fluorescence loss is analyzed in parental and MBD4Δ/Δ.14, MBD4Δ/Δ.11, and MBD4Δ/Δ.12 deletion clones by flow cytometry. Cells were FACS sorted at day 0 for IgM+GFP+ cells and re-analyzed after 28 days in culture. Flow cytometry analyses are representative of 24 parental- and 12 subclones from each of the MBD4Δ/Δ deletion clones. (D) IgM fluorescence loss was quantitated for each parental and MBD4Δ/Δ deletion subclone after 28 days in culture. Each data point is representative of a single sub-clone. Bars indicate the median in each data set. P-value *p < 0.05 and ***p < 0.0005 was analyzed using Kruskal-Wallis test.
Figure 4
Figure 4
SHMs occur largely at G/C residues in the IgL gene of MBD4Δ/Δ subclones. The leader and CDR1, CDR2 and CDR3 motifs in the reference sequence are boxed. Mutations are from the parental (n = 11, black; above the reference sequence) and MBD4Δ/Δ.11 (n = 8, green), MBD4Δ/Δ.12 (n = 12, blue), and MBD4Δ/Δ.14 (n = 12, red) deletion clones, below the reference sequence. The asterisk indicates that the mutated nucleotide sequence was mixed with the parental sequence at that position. AID hotspot motifs (RGYW, WRCY) are highlighted in bold. Nucleotide numbering is indicated at the end of each line.
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