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Cited by 3Genomics and Chromatin DynamicsDNA Repair MechanismsT-cell and B-cell Immunology

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Activation-induced cytidine deaminase an antibody diversification enzyme interacts with chromatin modifier UBN1 in B-cells

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Jaiswal, Ankit, Roy, Rajarshi, Tamrakar, Anubhav, Singh, Amit Kumar, Kar, Parimal, Kodgire, Prashant·2023·Scientific Reports
DOI 10.1038/s41598-023-46448-7PMC PMC10638239

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Abstract

Activation-induced cytidine deaminase (AID) is the key mediator of antibody diversification in activated B-cells by the process of somatic hypermutation (SHM) and class switch recombination (CSR). Targeting AID to the Ig genes requires transcription (initiation and elongation), enhancers, and its interaction with numerous factors. Furthermore, the HIRA chaperon complex, a regulator of chromatin architecture, is indispensable for SHM. The HIRA chaperon complex consists of UBN1, ASF1a, HIRA, and CABIN1 that deposit H3.3 onto the DNA, the SHM hallmark. We explored whether UBN1 interacts with AID using computational and in-vitro experiments. Interestingly, our in-silico studies, such as molecular docking and molecular dynamics simulation results, predict that AID interacts with UBN1. Subsequently, co-immunoprecipitation and pull-down experiments established interactions between UBN1 and AID inside B-cells. Additionally, a double immunofluorescence assay confirmed that AID and UBN1 were co-localized in the human and chicken B-cell lines. Moreover, proximity ligation assay studies validated that AID interacts with UBN1. Ours is the first report on the interaction of genome mutator enzyme AID with UBN1. Nevertheless, the fate of interaction between UBN1 and AID is yet to be explored in the context of SHM or CSR.

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Figures

Figure 1 (A) The structural domain of human AID. (B) The structural domain of UBN1. The NHRD domain interacts with HIRA, and the HRD domain interacts with H3.3/H4. (C) UBN1 structure predicted by I-TASSER. (D) AID structure as predicted by alphafold2 (AF-Q9GZX7-F1).
Figure 1 (A) The structural domain of human AID. (B) The structural domain of UBN1. The NHRD domain interacts with HIRA, and the HRD domain interacts with H3.3/H4. (C) UBN1 structure predicted by I-TASSER. (D) AID structure as predicted by alphafold2 (AF-Q9GZX7-F1).CC BY · PubMed Central
Figure 2 (A) UBN1 and AID complex after docking, (B) UBN1 and AID complex after MD simulation. UBN1: Violet, AID: Green, (C) Binding free energy and its component for UBN1 and AID complex, (D) Residue-wise contribution of binding free energy. Key residues are mentioned in the plot.
Figure 2 (A) UBN1 and AID complex after docking, (B) UBN1 and AID complex after MD simulation. UBN1: Violet, AID: Green, (C) Binding free energy and its component for UBN1 and AID complex, (D) Residue-wise contribution of binding free energy. Key residues are mentioned in the plot.CC BY · PubMed Central
Figure 3 AID interaction with UBN1 in Raji cells. (A) Co-immunoprecipitation of UBN1 from whole cell lysate was performed from Raji cells using anti-UBN1 pAb and analyzed by western blotting using protein anti-AID mAb and anti-UBN1 pAb. (B) Co-immunoprecipitation of AID from whole cell lysate of Raji cells using anti-AID mAb and analyzed by western blotting using anti-UBN1 pAB and anti-AID mAb.
Figure 3 AID interaction with UBN1 in Raji cells. (A) Co-immunoprecipitation of UBN1 from whole cell lysate was performed from Raji cells using anti-UBN1 pAb and analyzed by western blotting using protein anti-AID mAb and anti-UBN1 pAb. (B) Co-immunoprecipitation of AID from whole cell lysate of Raji cells using anti-AID mAb and analyzed by western blotting using anti-UBN1 pAB and anti-AID mAb.CC BY · PubMed Central
Figure 4 AID interaction with UBN1 in DT40ψV KO cells. (A) Co-immunoprecipitation of UBN1 from whole cell lysate was performed from DT40ψV KO using anti-UBN1 pAb and analyzed by western blotting using protein anti-AID mAb and anti-UBN1 pAb. (B) Co-immunoprecipitation of AID from whole cell lysate of DT40ψV KO cells using anti-AID mAb and analyzed by western blotting using anti-UBN1 pAB and anti-AID mAb.
Figure 4 AID interaction with UBN1 in DT40ψV KO cells. (A) Co-immunoprecipitation of UBN1 from whole cell lysate was performed from DT40ψV KO using anti-UBN1 pAb and analyzed by western blotting using protein anti-AID mAb and anti-UBN1 pAb. (B) Co-immunoprecipitation of AID from whole cell lysate of DT40ψV KO cells using anti-AID mAb and analyzed by western blotting using anti-UBN1 pAB and anti-AID mAb.CC BY · PubMed Central
Figure 5 His-tagged pull-down assay for AID interaction with UBN1 in Raji and DT40ψV KO cells. (A) His tagged MBP-AID or MBP were immobilized on Ni-NTA beads followed by incubation with Raji cell lysate and analysed by western blotting using anti-UBN1 pAb. (B) His tagged MBP-UBN1 or MBP were immobilized on Ni-NTA beads followed by incubation with Raji cell lysate and analysed by western blotting using anti-AID mAb. (C) His tagged MBP-AID or MBP were immobilized on Ni-NTA beads followed by incubation with DT40ψV KO cell lysate and analysed by western blotting using anti-UBN1 pAB. (D) His tagged MBP-UBN1 or MBP were immobilized on Ni-NTA beads followed by incubation with DT40ψV KO cell lysate and analysed by western blotting using anti-AID mAB.
Figure 5 His-tagged pull-down assay for AID interaction with UBN1 in Raji and DT40ψV KO cells. (A) His tagged MBP-AID or MBP were immobilized on Ni-NTA beads followed by incubation with Raji cell lysate and analysed by western blotting using anti-UBN1 pAb. (B) His tagged MBP-UBN1 or MBP were immobilized on Ni-NTA beads followed by incubation with Raji cell lysate and analysed by western blotting using anti-AID mAb. (C) His tagged MBP-AID or MBP were immobilized on Ni-NTA beads followed by incubation with DT40ψV KO cell lysate and analysed by western blotting using anti-UBN1 pAB. (D) His tagged MBP-UBN1 or MBP were immobilized on Ni-NTA beads followed by incubation with DT40ψV KO cell lysate and analysed by western blotting using anti-AID mAB.CC BY · PubMed Central
Figure 6 In-vitro pull-down assay using purified protein to detect the interaction between UBN1 and hAID. (A, B) represent the strategy used for pull-down in (C, D), respectively. (C) MBP-hAID (w/o His tag) was added to His-MBP-Flag-UBN11-175 and His-MBP, followed by pull-down using Ni-NTA beads and detected by western blotting using anti-AID mAb. (D) MBP-Flag-UBN11-175 (w/o His-tag) was added to His-MBP-hAID and His-MBP, followed by pull-down using Ni-NTA beads and detection by western blotting using anti-Flag-mAb.
Figure 6 In-vitro pull-down assay using purified protein to detect the interaction between UBN1 and hAID. (A, B) represent the strategy used for pull-down in (C, D), respectively. (C) MBP-hAID (w/o His tag) was added to His-MBP-Flag-UBN11-175 and His-MBP, followed by pull-down using Ni-NTA beads and detected by western blotting using anti-AID mAb. (D) MBP-Flag-UBN11-175 (w/o His-tag) was added to His-MBP-hAID and His-MBP, followed by pull-down using Ni-NTA beads and detection by western blotting using anti-Flag-mAb.CC BY · PubMed Central
Figure 7 Co-localization of AID with UBN1 in DT40ѰV KO cells. Cells were fixed and double stained with monoclonal anti-AID (green) and polyclonal anti-UBN1 (red) antibodies. AID is predominantly located in the cytoplasm, whereas UBN1 is mostly inside the nucleus. Yellow and orange spots are produced due to the co-localization of AID with UBN1.
Figure 7 Co-localization of AID with UBN1 in DT40ѰV KO cells. Cells were fixed and double stained with monoclonal anti-AID (green) and polyclonal anti-UBN1 (red) antibodies. AID is predominantly located in the cytoplasm, whereas UBN1 is mostly inside the nucleus. Yellow and orange spots are produced due to the co-localization of AID with UBN1.CC BY · PubMed Central
Figure 8 Co-localization of AID with UBN1 in Raji cells. Cells were fixed and double stained with monoclonal anti-AID (green) and polyclonal anti-UBN1 (red) antibodies. AID is predominantly located in the cytoplasm, whereas UBN1 is mostly inside the nucleus. Yellow and orange spots are produced due to the co-localization of AID with UBN1.
Figure 8 Co-localization of AID with UBN1 in Raji cells. Cells were fixed and double stained with monoclonal anti-AID (green) and polyclonal anti-UBN1 (red) antibodies. AID is predominantly located in the cytoplasm, whereas UBN1 is mostly inside the nucleus. Yellow and orange spots are produced due to the co-localization of AID with UBN1.CC BY · PubMed Central
Figure 9 Proximity ligation assay for AID and UBN1 in Raji cells. Images demonstrate the PLA signals in Raji cells and the proximity between two proteins, AID and UBN1. Nuclei were stained with DAPI. Anti-AID and anti-UBN1 primary antibodies were incubated, followed by incubation with secondary antibodies conjugated to oligonucleotides (PLA Probes anti-mouse MINUS and PLA Probes anti-rabbit PLUS). Red spots in the cells shown by the arrow indicate the interaction between UBN1 and AID.
Figure 9 Proximity ligation assay for AID and UBN1 in Raji cells. Images demonstrate the PLA signals in Raji cells and the proximity between two proteins, AID and UBN1. Nuclei were stained with DAPI. Anti-AID and anti-UBN1 primary antibodies were incubated, followed by incubation with secondary antibodies conjugated to oligonucleotides (PLA Probes anti-mouse MINUS and PLA Probes anti-rabbit PLUS). Red spots in the cells shown by the arrow indicate the interaction between UBN1 and AID.CC BY · PubMed Central
Figure 10 Proximity ligation assay for AID and UBN1 in DT40ψV KO cells. Images demonstrate the PLA signals in DT40ψV KO and the proximity between two proteins, AID and UBN1. Nuclei were stained with DAPI. Anti-AID and anti-UBN1 primary antibodies were incubated, followed by incubation with secondary antibodies conjugated to oligonucleotides (PLA Probes anti-mouse MINUS and PLA Probes anti-rabbit PLUS). Red spots in the cells shown by the arrow indicate the interaction between UBN1 and AID.
Figure 10 Proximity ligation assay for AID and UBN1 in DT40ψV KO cells. Images demonstrate the PLA signals in DT40ψV KO and the proximity between two proteins, AID and UBN1. Nuclei were stained with DAPI. Anti-AID and anti-UBN1 primary antibodies were incubated, followed by incubation with secondary antibodies conjugated to oligonucleotides (PLA Probes anti-mouse MINUS and PLA Probes anti-rabbit PLUS). Red spots in the cells shown by the arrow indicate the interaction between UBN1 and AID.CC BY · PubMed Central
Figure 11 UBN1 interacts with AID as well as H3.3. Due to this interaction, UBN1 may be able to load H3.3 and AID in complex forms to the variable region of the Ig locus.
Figure 11 UBN1 interacts with AID as well as H3.3. Due to this interaction, UBN1 may be able to load H3.3 and AID in complex forms to the variable region of the Ig locus.CC BY · PubMed Central

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Research works printed but not linked: 1

  1. Kräutler, V., van Gunsteren, W. F. & Hünenberger, P. H. A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J. Comput. Chem. 22(5), 501–508. https://doi.org/10.1002/1096-987X(20010415)22:5%3C501::AID-JCC1021%3E3.0.CO (2001). · DOI

Metadata

Authors
Jaiswal, A., Roy, R., Tamrakar, A., Singh, A. K., Kar, P., & Kodgire, P.
Affiliations
Indian Institute of Technology Indore (IN)
Corresponding author
Kodgire, Prashant
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2023-11-10
Volume
13
Issue
1
Pages
19615-19615
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-023-46448-7
PMC
PMC10638239
OpenAlex
W4388573268
Cited by
3
References
51
Citations by year
2026: 2 · 2025: 1
FWCI
0.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Science and Engineering Research Board (CRG/2020/358)
  • Indian Council of Medical Research (OMI/17/2020-ECD-1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords
activation-induced cytidine deaminase, antibody diversification, somatic hypermutation, class switch recombination, B cells, protein-protein interaction, chromatin modification, immunoglobulin genes
MeSH terms
AICDA (Activation-Induced Cytidine Deaminase), Chromatin, Cytidine Deaminase, Humans, Immunoglobulins, Nuclear Proteins, Transcription Factors, Immunoglobulin Class Switching, Molecular Chaperones, Somatic Hypermutation, Immunoglobulin, Molecular Docking Simulation

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