Immunoglobulin Gene Rearrangement Alison Brittain Mahboubeh Noori Noora

Immunoglobulin Gene Rearrangement Alison Brittain Mahboubeh Noori (Noora) Christian Showalter MCB 7200 Dr. Horodyski 1

Outline: • Introduction to immunoglobulins and V(D)J recombination • Mechanism of V(D)J recombination • Errors in V(D)J recombination 2

Immunoglobulins (Ig’s): • Secreted by B cells and act as B cell receptors (BCR) • Binds antigens = any substance eliciting an immune response 3

Role of Immunoglobulins (Ig’s) in Antigen Recognition: • Appropriate Ig’s are secreted by B cells after they differentiate into plasma cells in the germinal center of a lymph node 4

Ig Isotypes: 5

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Ig Structure and Variability in Antigen Recognition – V(D)J Segments: 7

V(D)J Recombination: • 44 variable, 27 diversity, 6 joining • 3× 1011 possible combinations 8

Rearrangement of Ig gene segments: http: //what-when-how. com/wp-content/uploads/2012/04/tmp 4 B 108. jpg 9

How Does Rearrangement Occur? • Rearrangement occurs between specific sites on the DNA called Recombination Signal Sequences (RSSs). • Rearrangement is catalyzed by two Recombination Activating Genes: RAG-1 and RAG-2. 10

Recombination Signal Sequence (RSS): • A short DNA sequence containing a conserved heptamer and nonamer separated by either 12 or 23 base pair, indicate the sites of recombination • Two types of RSS exist • Each V, D, or J gene segment is flanked by RSS • The RSSs are present on the 3’ side (downstream) of a V region and the 5’ side (upstream) of the J region V, D, or j coding flank Consensus 12 or 23 -RSS 12 or 23 on binati m o c e r f o 2/23 rule the 1 11 Schatz, David G. , and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation. " Annual review of genetics 45 (2011): 167 -202.

Recombination Activating Gene (RAG): 12 Schatz, David G. , and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation. " Annual review of genetics 45 (2011): 167 -202.

V(D)J recombination: Nicking: first step of DNA cleavage by RAG in which one DNA strand is broken 5’ of the heptamer Hairpin formation: second step of DNA cleavage by RAG in which the 3’-hydroxyl of the nicked strand attacks the other strand HMGB: high mobility group box Paired (synaptic) complex (PC): protein-DNA complex in which the two RSSs are held in close juxtaposition by the RAG proteins Signal end: after DNA cleavage by the RAG proteins, the DNA end that terminates in the RSS Coding end: after DNA cleavage by the RAG proteins, the DNA end that terminates in the coding segment CSC: cleaved signal complex SEC: signal end complex Nonhomologous end joining (NHEJ): a DNA repair process that joins broken DNA ends (double-strand breaks) without using homologous DNA as a template 13 Schatz, David G. , and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation. " Annual review of genetics 45 (2011): 167 -202.

V(D)J recombination: RSS Rag-1 + Rag-2 DNA-PK Artemis Ku DNA ligase/XRCC 4 dimer https: //www. youtube. com/watch? v=QTOBSFJWog. E 14

Products of V(D)J recombination: RSSs relative orientation determines whether the reaction proceeds by inversion or by deletion Inappropriate joining of a coding end to an RSS Roth, David B. "V (D) J Recombination: Mechanism, Errors, and Fidelity. "Microbiology Spectrum 2. 6 (2014). 15

V(D)J Recombination Errors: • The rearranging of genes to generate the large amount of antigen-receptor diversity is prone to errors. • Deleterious genomic rearrangements can be potentially created as a result of V(D)J recombination. • Chromosome translocations can result in cancerous growths, such as in lymphomas and leukemia, as a result of kilo base to mega base inversions and deletions. 16

V(D)J Recombination Errors: • There are two main categories of errors as a result of V(D)J recombination 1. Errors in Target Recognition 2. Errors in End Joining 17

Errors in Target Recognition: • When errors in V(D)J recombination occur in lymphoid neoplasms it may result in deleterious chromosomal rearrangements • First type of error is due to when an RSS and a DNA sequence resembling RSS known as “cryptic RSS” are recognized • Small size of RSS sequences and the fact that recombination does not necessitate strict adherence of consensus heptane/monomer sequences allows for c. RSSs capable of supporting recombination about once per kilobase in random DNA sequences • Roth D. 2014. V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: 10. 1128/microbiolspec. MDNA 3 -0041 -2014 Analysis of human lymphoid neoplasms have revealed chromosome translocations involving mismatching of RSSs and c. RSSs that are adjacent to proto-oncogenes 18

Errors in Target Recognition Continued: • Errors in mismatched RSSs and c. RSSs can cause inappropriate expression of a target gene due to presence of a transcriptional regulator on the antigen-receptor loci • Pairs of c. RSSs can create chromosome translocations such as in T-cell acute lymphoblastic leukemia cases involving translocations between T-cell receptor gene segments and the SCL locus in trans • Pairs of c. RSSs can create chromosome translocation events in cis by generating a deleted coding joint and an excised signal joint Roth D. 2014. V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: 10. 1128/microbiolspec. MDNA 3 -0041 -2014 19

Errors in Joining: • Errors in joining involve events that join a RAG-mediated double-strand break to a broken DNA end created by a non. RAG-mediated mechanism • A pair of breaks created during normal V(D)J recombination are mistakenly joined to another break created by another mechanism • Chromosome translocations or insertions of signal-ended fragments into another chromosomal location can occur Roth D. 2014. V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: 10. 1128/microbiolspec. MDNA 3 -0041 -2014 20

Chromosomal Rearrangements Cause B- and T-cell Neoplasms: • Neoplasms are new and abnormal growths of tissue on the body • Lymphomas are malignant growths of lymphatic tissue where lymphocytes are produced • A Chromosomal rearrangement as a result of V(D)J recombination can lead to may types of non. Hodgkin lymphomas such as follicular lymphoma, Burkitt’s lymphoma, and Chronic Myelogenous Leukemia Blausen. com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI: 10. 15347/wjm/2014. 010. ISSN 20018762. 21

Diseases Associated with Errors in V(D)J Recombination: • Burkitt’s lymphoma is a cancer of mostly B lymphocytes. C-myc is moved from its usual position on chromosome 8 to a location close to enhancers of the antibody heavy chain genes on chromosome 14 and its transcription is significantly increased. C-myc is a transcription factor playing a role in mitosis of mammalian cells which produces cancerous lymphocytes. • CML is a form of leukemia characterized by the increased and upregulated growth of predominately myeloid cells in the bone marrow and accumulation of them in the blood as a result of constitutive tyrosine kinase activity that activates a cascade of proteins that control the cell cycle and speeds up cell division. The BCR-ABL protein is also known to inhibit DNA repair 22

Regulation of V(D)J Recombination: • Recombinase is active only in the appropriate lymphocytes at specific times in development • RAG 1 and RAG 2 are carefully limited in cells and in stages of development • Another control is cell-cycle-specific protein degradation of the RAG 2 protein mediated by threonine phosphorylation (T 490) • Autoubiquitylation of RAG 1 may also play a role in regulating V(D)J recombination and preventing errors 23

Summary • V(D)J Recombination is a very useful mechanism to create an almost limitless supply of different antibodies to target antigens associated with pathogens, however, this mechanism can have errors associated with it as a result of the double stranded breaks in DNA which may sometimes lead to cancerous cells 24

References • Schatz, David G. , and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation. " Annual review of genetics 45 (2011): 167 -202 • https: //www. youtube. com/watch? v=QTOBSFJWog. E • http: //what-when-how. com/wp-content/uploads/2012/04/tmp 4 B 108. jpg • Roth D. 2014. V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: 10. 1128/microbiolspec. MDNA 3 -0041 -2014 • Blausen. com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI: 10. 15347/wjm/2014. 010. ISSN 20018762. • http: //image. slidesharecdn. com/neoplasia-robbinspath-120704091051 phpapp 02/95/neoplasia-robbins-path-33 -728. jpg? cb=1341393120 25

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