Immunoglobulin Gene Rearrangement Catherine Baugher Peifeng Yu

Presentation Overview Introduction to Immunoglobulin Gene Recombination Mechanism of V(D)J Recombiniation Errors in V(D)J Recombination

Immunoglobulin A large Y-shaped protein which has a site for binding of antigens Secreted by B lymphocytes to recognize and neutralize invading pathogens Each cell produces its own species of antibody with a unique binding site Once a B cell is activated by binding to the immunoglobulin, that species of antibody is secreted into the bloodstream. From Alberts B, Johnson A, Lewis J, et al. (2002) Molecular Biology of the Cell. 4th edition. Fig 24-17 When a “naive” B cell is activated by the antigen binding to it, it proliferates and differentiates into a “plasma B cell” which secretes more antibodies into the bloodstream. In order to account for the many different types of pathogens in existence, the antibody must differ from cell-to-cell. Because of this, there is a variable region of the antibody. (leads into next slide)

Isotypes of Immunoglobulin IgG - Secondary immune response IgM - Primary immune response IgA - Mucous membranes, has a secretory component IgD - Precise function unknown, shares IgM’s role IgE - Allergic reaction, parasite immunity Isotype is defined by a constant region of the protein From Absolute Antibody (2014). https://absoluteantibody.com/wp-content/uploads/2014/03/isotypes.png IgG, IgD and IgE are examples of monomers, while the other isotypes are made up of multiple Y-structures. There are five main types of immunoglobulin in humans and their primary functions. Listed in order of their concentration, e.g. IgG makes up most of the immunoglobulin in the body (70-85%). IgA has a secretory component which allows it to be transported across epithelial cells, as well as protects it from digestion in the stomach. IgE might play a role in parasite immunity, but it’s most well-documented for causing allergic reactions-- histamine response and possibly anaphylaxis. On the right is a preview into the structure of immunoglobulin, and there is a constant region called Fc which the isotype defines, but there’s also a variable region called Fab which allows for different antigen binding sites (leads into next slide)

Protein Structure The heavy chain is primarily constant, apart from the area of antigen binding. Defines the Isotype. Heavy chains connected with a disulfide bonds Light chain is smaller and also has a variable region Heavy and light chains are linked by disulfide bonds From Thermo Fisher Scientific (2014). https://www.thermofisher.com/us/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/immunoglobulin-structure-classes.html How can the antibodies differ from cell-to-cell if the same genetic code is being used? Isotype is defined by the constant region, or lower half of the Y, is heavy chain, and connected with disulfide bonds There is a variable region which allows for different antigen binding sites. The question then arises, how does this variable region come about if the genetic code to produce them is the same?

Immunoglobulin Development B cells vary in structure and genetic makeup during development Prior to antigen binding, genetic rearrangements occur to develop a unique antigen binding site Early B cells undergo genetic rearrangements called V(D)J rearrangements From Mulligan S. (1989) Leukemia & Lymphoma. Fig 1

Gene segments in Immunoglobulin Coding DNA V - Variable segments D - Diversity segments J - Joining segments From Pelsue S. (2016) Marine Biotechnology. Fig 1 Random rearrangements of these segments results in millions of different combinations of antibodies = V(D)J recombination Heavy chains have V, D and J regions. Light chains only have V and J.

Genetic Recombination of Heavy chain DNA between D and J segments deleted, brought together V segment joins the DJ complex Pre-mRNA is transcribed Sequence between VDJ complex and constant segments removed, poly-A tail added mRNA translated to produce heavy chain From gustavocarra. (2008) distributed under a CC-BY 2.0 license.

Genetic Recombination of Light chain Light chains lack D segments So V and J combine to form a VJ complex From Hunt R. University of South Carolina School of Medicine http://www.microbiologybook.org/mayer/gen-1.jpg Light chain recombination is very similar to heavy chain recombination, however D segments are absent

V(D)J recombination First, a D and a J segment are chosen from among several possibilities, and are brought together to form a D-J rearrangement. Then a V region is selected and joined with the D-J rearrangement to form a complete VDJ exon From David B. Roth et al., (2014) Microbiol Spectr.

Steps in V(D)J Recombination 12-RSS or 23-RSS are assembled into a synaptic complex by the RAG proteins, possibly assisted by HMGB1/2 Signal and coding joint formation is mediated by the NHEJ pathway, which includes Ku70, Ku80, XRCC4, DNA Ligase IV and Cernunnos (XLF). From Yanhong et al ., Cell. 3, 141 (2010)

The RAG proteins Each RAG protein have been defined as the minimal portion of the protein required for V(D)J recombination activity The RAG2 core is crucial for DNA cleavage activity From David G. Schatz W,. (2011) Nature Reviews Immunology.

V(D)J recombination: RAG1 and RAG2 binding. Formation of double-stranded break From Immunopaedia.org

V(D)J recombination: Non-homologous end-joining: Recognition and end binding End processing From Immunopaedia.org

V(D)J recombination: Non-homologous end-joining: DNA polymerization DNA ligation From Immunopaedia.org

Mechanism of V(D)J recombination In the first phase, RAG1 and RAG2 proteins bind to 12RSS or the 23RSS. In the second phage, the RAG proteins coorperate with NHEJ DNA repair factors to rejoin the DNA end. From David G. Schatz W,. (2011) Nature Reviews Immunology.

Recombination Errors: Target Recognition RSS is simply a consensus sequence, so sequences similar to RSS (cRSS) may be wrongfully targeted. Erroneous deletion of region between RSS-like regions RSS binds to form a mismatched RSS/cRSS recognition of one authentic RSS and one DNA sequence fortuitously resembling an RSS (termed a “cryptic RSS” or cRSS) Small size of RSS sequences and does not require strict adherence of this sequence to consensus heptamer/nonamer sequences, so randomly searching in DNA yields 1 cRSS per kilobase unit Mutation can lead to leukemia (if adjacent to proto oncogene) In a, authentic RSS pairs w cRSS to form a pseudo coding joint and a pseudo signal joint In b, a pair of cRSS located on separate DNA molecules join, generating a chromosome translocation In c, two cRSS are recombined again, but on the same chromosome, causing deletions in the DNA Roth, D. (2014). Microbiol Spectr, 2(6).

Recombination Errors: Joining Between normal V(D)J with an authentic RSS and a chromosome break from unrelated means Pair of cRSS end joining Can have a combination of recognition and joining errors: cleavage at a pair of cRSS followed by joining to a non-RAG mediated DSB Can also cause chromosomal translocation events Roth, D. (2014). Microbiol Spectr, 2(6). A pair of breaks created during normal V(D)J recombination are mistakenly joined to another break created by another mechanism Can occur between an authentic RSS and a chromosome break from an unrelated means, or between a pair of cRSS 1. In a, a normal end donation

Genetic anomalies due to errors Lymphoid neoplasm – proliferation of B- and T-lymphocytes Causes lymphoma and lymphoid leukemia Can cause expression of a gene due to presence of a transcriptional regulatory elements from the antigen receptor loci Thus, oncogenic events can occur Some systems are in place to ensure fidelity Main thing it causes is Lymphoid neoplasm because we’re dealing with lymphocytes, and this is actually what spurred on research into V(D)J errors. For example (to last point) ensuring that the recombinase is active only in the appropriate target cells, and only during the appropriate developmental stages reduces error because RAG1 and RAG2 are expressed in a carefully limited in a cell- and developmental stage-specific fashion From Cancer Research UK. (2014) distributed under a CC-BY 4.0 license.

Summary Genetic variation occurs because of recombination Recombination may occur between the light and heavy chains to create unique antibodies for millions of potential antigens Non-Homologous End Joining pathways provide a mechanism of action Mutations in this mechanism lead to errors in target recognition and joining errors, potentially leading to cancer development

Resources Lee, G.S, et al. (2004). RAG Proteins Shepherd Double-Strand Breaks to a Specific Pathway, Suppressing Error-Prone Repair, but RAG Nicking Initiates Homologous Recombination. Cell, 117, 171-184. Qiu, J, et al. (2001). Separation-of-Function Mutants Reveal Critical Roles for RAG2 in Both the Cleavage and Joining Steps of V(D)J Recombination. Molecular Cell, 7, 77-87. Roth D. B. (2014). V(D)J Recombination: Mechanism, Errors, and Fidelity. Microbiology spectrum, 2(6), 10.1128/microbiolspec.MDNA3-0041-2014. Schatz, D. G., & Swanson, P. (2011). V(D)J recombination: Mechanisms of initiation. Annual Review of Genetics, 45, 167-202. https://doi.org/10.1146/annurev-genet-110410-132552 Stephen P. Mulligan (1990) Human B Cells: Differentiation and Neoplasia, Leukemia & Lymphoma,1:5-6, 275-291, DOI: 10.1080/10428199009169597 Yanhong Ji, David G. Schatz. (2010) The In Vivo Pattern of Binding of RAG1 and RAG2 to Antigen Receptor Loci. Cell, 141, 419-431. https://www.sciencedirect.com/science/article/pii/S0092867410002461#fig8