Similar Datasets
Project description:Nuclear lamina dynamic changes influence chromatin landscape. Mechanisms governing nuclear lamina behavior have not been studied within the adaptive immune context. Here, we show autophagy regulates withdrawal of nuclear lamina protein, Lamin B1, in B cells transiting germinal centers (GCs). Autophagy inhibition was sufficient to keep Lamin B1 nuclear integrity preventing NP-CGG immunisation-induced nucleotide substitution during the somatic hypermutation process resulting in compromised B cell capacity to build up a competitive high-affinity antibodies. Atg7 loss-of-function in GC B cells resolved in decreased chromatin accessibility. Single-cell RNAseq revealed centroblasts accumulate in dark zone upon autophagy impairment due to inefficient signals to finish GC reaction. Hence, GC B cells rely on autophagy for Lamin B1 disassembly, becoming autophagy key part of somatic hypermutation process and centroblast dynamics.
2025-05-12 | GSE218051 | GEO
Project description:Nuclear lamina dynamic changes influence chromatin landscape. Mechanisms governing nuclear lamina behavior have not been studied within the adaptive immune context. Here, we show autophagy regulates withdrawal of nuclear lamina protein, Lamin B1, in B cells transiting germinal centers (GCs). Autophagy inhibition was sufficient to keep Lamin B1 nuclear integrity preventing NP-CGG immunisation-induced nucleotide substitution during the somatic hypermutation process resulting in compromised B cell capacity to build up a competitive high-affinity antibodies. Atg7 loss-of-function in GC B cells resolved in decreased chromatin accessibility. Single-cell RNAseq revealed centroblasts accumulate in dark zone upon autophagy impairment due to inefficient signals to finish GC reaction. Hence, GC B cells rely on autophagy for Lamin B1 disassembly, becoming autophagy key part of somatic hypermutation process and centroblast dynamics.
2025-05-12 | GSE218050 | GEO
Project description:Autophagy controls DNA accessibility to immunoglobulin variable regions and centroblast dynamics
| PRJNA901988 | ENA
Project description:Autophagy controls DNA accessibility to immunoglobulin variable regions and centroblast dynamics [scRNA-seq]
| PRJNA901991 | ENA
Project description:Autophagy controls DNA accessibility to immunoglobulin variable regions and centroblast dynamics [scATAC-seq]
| PRJNA901994 | ENA
Project description:Low-fidelity DNA polymerases introduce nucleotide substitutions in immunoglobulin variable regions during somatic hypermutation. Although DNA polymerase (pol) η is the major low-fidelity polymerase, other DNA polymerases may also contribute. Existing data are contradictory as to whether pol ζ is involved. We reasoned that the presence of pol η may mask the contribution of pol ζ, and therefore we generated mice deficient for pol η and heterozygous for pol ζ. The frequency and spectra of hypermutation was unaltered between Polζ(+/-) Polη(-/-) and Polζ(+/+) Polη(-/-) clones. However, there was a decrease in tandem double-base substitutions in Polζ(+/-) Polη(-/-) cells compared with Polζ(+/+) Polη(-/-) cells, suggesting that pol ζ generates tandem mutations. Contiguous mutations are consistent with the biochemical property of pol ζ to extend a mismatch with a second mutation. The presence of this unique signature implies that pol ζ contributes to mutational synthesis in vivo. Additionally, data on tandem mutations from wild type, Polζ(+/-), Polζ(-/-), Ung(-/-), Msh2(-/-), Msh6(-/-), and Ung(-/-) Msh2(-/-) clones suggest that pol ζ may function in the MSH2-MSH6 pathway.
| S-EPMC3371727 | biostudies-literature
Project description:The humoral immune response plays a critical role in controlling infection, and the rapid adaptation to a broad range of pathogens depends on a highly diverse antibody repertoire. The advent of high-throughput sequencing technologies in the past decade has enabled insights into this immense diversity. However, not only the variable, but also the constant region of antibodies determines their in vivo activity. Antibody isotypes differ in effector functions and are thought to play a defining role in elicitation of immune responses, both in natural infection and in vaccination. We have developed an Illumina MiSeq high-throughput sequencing protocol that allows determination of the human IgG subtype alongside sequencing full-length antibody variable heavy chain regions. We thereby took advantage of the Illumina procedure containing two additional short reads as identifiers. By performing paired-end sequencing of the variable regions and customizing one of the identifier sequences to distinguish IgG subtypes, IgG transcripts with linked information of variable regions and IgG subtype can be retrieved. We applied our new method to the analysis of the IgG variable region repertoire from PBMC of an HIV-1 infected individual confirmed to have serum antibody reactivity to the Membrane Proximal External Region (MPER) of gp41. We found that IgG3 subtype frequencies in the memory B cell compartment increased after halted treatment and coincided with increased plasma antibody reactivity against the MPER domain. The sequencing strategy we developed is not restricted to analysis of IgG. It can be adopted for any Ig subtyping and beyond that for any research question where phasing of distant regions on the same amplicon is needed.
| S-EPMC4218849 | biostudies-literature
Project description:Germline variations in immunoglobulin genes influence the repertoire of B cell receptors and antibodies, and such polymorphisms may impact disease susceptibility. However, the knowledge of the genomic variation of the immunoglobulin loci is scarce. Here, we report 25 potential novel germline IGHV alleles as inferred from rearranged naïve B cell cDNA repertoires of 98 individuals. Thirteen novel alleles were selected for validation, out of which ten were successfully confirmed by targeted amplification and Sanger sequencing of non-B cell DNA. Moreover, we detected a high degree of variability upstream of the V-REGION in the 5'UTR, L-PART1 and L-PART2 sequences, and found that identical V-REGION alleles can differ in upstream sequences. Thus, we have identified a large genetic variation not only in the V-REGION but also in the upstream sequences of IGHV genes. Our findings provide a new perspective for annotating immunoglobulin repertoire sequencing data.
| S-EPMC7261178 | biostudies-literature
Project description:Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-β-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring β-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-β-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-β-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.
| S-EPMC10641266 | biostudies-literature
Project description:Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2-hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry-exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.
| S-EPMC3488218 | biostudies-literature