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

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

3. Immunoglobulins (Ig’s):
Ig’s contain a variable (V) and constant (C) region, the variable being the region that binds the antigen. They also contain light and heavy chains, the heavy chains being the anchor into cell surfaces. Light chains are encoded on chromosomes 2 and 22, heavy chains on chromosome 17.
Secreted by B cells and act as B cell receptors (BCR)
Binds antigens = any substance eliciting an immune response

4. 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
Ig’s are secreted from matured B cells, which are then called plasma cells.
Antigens are recognized by antigen-presenting cells, some of which are T cells or other B cells. When attached to their receptors, TCRs communicate with BCR’s, or BCRs induce other B cells with the correct antibody for the antigen, to proliferate and produce massive quantities of antibodies specific for the antigen.
Free-floating antigens can recognize small antigens or large ones (like parasites) and perform “opsonization”, which draws more immune cells to the area (like macrophages and monocytes) that can digest larger antigens.

5. Ig Isotypes:
Ig’s come in different forms depending upon the antigen that a mature B cell recognizes- it will first produce IgM, then after being triggered by an antigen-presenting cell (via its BCR surface Ig) and in the presence of specific cytokines, it can swtich to other classes. Different classes do different things: IgA is important for mucosal immunity, IgM is the largest and first formed by any B cell, IgG is a memory Ig and is the only Ig that can cross the placenta, IgD is essentially the BCR, IgE can cause mast cell degranulation in an allergic response

6. OVERVIEW SLIDE: VDJ recombination occurs in the bone marrow in newly synthesized B cells, this imparts specificity for their antigen-recognition sites
The immature B cell then leaves the bone marrow and awaits antigen presentation in the germinal center of a lymph node
Once the appropriate antigen is presented to the B cell by a T cell or other APC, the B cell with the appropriate antibodies begins massively replicating and secreting loads of antibodies as plasma cells. This stage has nothing to do with VDJ recombination.
From there, a subset of B cells retain memory and live forever with the capacity to secrete high amounts of antibodies the next time it encounters that same antigen.

7. Ig Structure and Variability in Antigen Recognition – V(D)J Segments:
Naïve B cells leave the bone marrow having already undergone VDJ recombination- thus exhibiting numerous variations in the structure of the variable regions of their Ig’s. Heavy chains have VDJ segments, whereas light chains only have VJ segments. These segments recombine randomly in the bone marrow to produce massive populations of B cells that have varying structures in their antigen-recognizing regions, thus each will recognize a different antigen. Once VDJ recombination occurs, they go to the germinal center and await activation by T cells. TCR’s also have VDJ/VJ recombination that imparts specificity of their receptors for certain antigens.

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

9. Rearrangement of Ig gene segments:

10. 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.
The RAGs encode enzymes that play an important role in the rearrangement and recombination of the genes of immunoglobulin and T cell receptor molecules during the process ofVDJ recombination

11. 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
12 or 23
Consensus 12 or 23 -RSS
V, D, or j coding flank
RSSs are recognized by a group of enzymes known collectively as the VDJ recombinase. RSSs are composed of seven conserved nucleotides (a heptamer) that reside next to the gene encoding sequence followed by a spacer (containing either 12 or 23 unconserved nucleotides) followed by a conserved nonamer (9 base pairs).
 spacer of 12 nucleotides will be recombined with one that has a spacer containing 23 nucleotides). This is known as the 12/23 rule of recombination (or the one-turn/two-turn rule)
the 12/23 rule of recombination
Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011):

12. Recombination Activating Gene (RAG):
 RAG proteins are fairly large. For example, mouse RAG-1 contains 1040 amino acids and mouse RAG-2 contains 527 amino acids.
The enzymatic activity of the RAG proteins is concentrated largely in a core region;
The RAG-1 core contains three acidic residues (D600, D708, and E962) in what is called the DDE motif, the major active site for DNA cleavage. These residues are critical for nicking the DNA strand and for forming the DNA hairpin
RAG enzymes work as a multi-subunit complex to induce cleavage of a single double stranded DNA (dsDNA) molecule between the antigen receptor coding segment and a flanking RSS.
Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011):

13. 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
They do this in two steps. They initially introduce a ‘nick’ in the 5' (upstream) end of the RSS heptamer (a conserved region of 7 nucleotides) that is adjacent to the coding sequence, leaving behind a specific biochemical structure on this region of DNA: a 3'-hydroxyl (OH) group at the coding end and a 5'-phosphate (PO4) group at the RSS end. The next step couples these chemical groups, binding the OH-group (on the coding end) to the PO4-group (that is sitting between the RSS and the gene segment on the opposite strand). This produces a 5'-phosphorylated double-stranded break at the RSS and a covalently closed hairpin at the coding end. The RAG proteins remain at these junctions until other enzymes (notably, TDT) repair the DNA breaks.
The RAG proteins initiate V(D)J recombination, which is essential for the maturation of pre-B and pre-T cells
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
Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011):

14. V(D)J recombination: RSS Rag-1 + Rag-2 Artemis DNA-PK
DNA ligase/XRCC4 dimer Ku

15. 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).

16. 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.

17. 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

18. 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 cRSSs capable of supporting recombination about once per kilobase in random DNA sequences
Analysis of human lymphoid neoplasms have revealed chromosome translocations involving mismatching of RSSs and cRSSs that are adjacent to proto-oncogenes
Roth D V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: /microbiolspec.MDNA

19. Errors in Target Recognition Continued:
Errors in mismatched RSSs and cRSSs can cause inappropriate expression of a target gene due to presence of a transcriptional regulator on the antigen-receptor loci
Pairs of cRSSs 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 cRSSs can create chromosome translocation events in cis by generating a deleted coding joint and an excised signal joint
Roth D V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: /microbiolspec.MDNA

20. 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 V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: /microbiolspec.MDNA

21. 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: /wjm/ ISSN

22. 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

23. Regulation of V(D)J Recombination:
Recombinase is active only in the appropriate lymphocytes at specific times in development
RAG1 and RAG2 are carefully limited in cells and in stages of development
Another control is cell-cycle-specific protein degradation of the RAG2 protein mediated by threonine phosphorylation (T490)
Autoubiquitylation of RAG1 may also play a role in regulating V(D)J recombination and preventing errors

24. 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

25. References
Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011):
Roth D V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi: /microbiolspec.MDNA
Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI: /wjm/ ISSN
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