immunoglobulin gene rearrangement alison brittain mahboubeh noori (noora) christian showalter mcb...

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Immunoglobulin Gene Rearrangement

Alison Brittain

Mahboubeh Noori (Noora)Christian Showalter

MCB 7200

Dr. Horodyski

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Outline:

• Introduction to immunoglobulins and V(D)J

recombination

• Mechanism of V(D)J recombination

• Errors in V(D)J recombination

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Immunoglobulins (Ig’s):

• Secreted by B cells and act as B cell receptors (BCR)• Binds antigens = any substance eliciting an immune

response

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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

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Ig Isotypes:

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

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V(D)J Recombination:

• 44 variable, 27 diversity, 6 joining• 3×1011 possible combinations

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Rearrangement of Ig gene segments:

http://what-when-how.com/wp-content/uploads/2012/04/tmp4B108.jpg

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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.

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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

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

the 12/23 rule of recombination

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Recombination Activating Gene (RAG):


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Nicking: first step of DNA cleavage by RAG in which one DNA strand is broken 5’ of the heptamer

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 segmentCSC: 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

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

V(D)J recombination:


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Artemis

DNA-PK

DNA ligase/XRCC4 dimer

Ku

RSS Rag-1 + Rag-2

V(D)J recombination:

https://www.youtube.com/watch?v=QTOBSFJWogE

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Products of V(D)J recombination:

Inappropriate joining of a coding end to an RSS

RSSs relative orientation determines whether the reaction proceeds by inversion or by

deletion

Roth, David B. "V (D) J Recombination: Mechanism, Errors, and Fidelity."Microbiology Spectrum 2.6 (2014).

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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.

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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

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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. 2014. V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi:10.1128/microbiolspec.MDNA3-0041-2014

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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


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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


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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.

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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

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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

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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

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• 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=QTOBSFJWogE

• http://what-when-how.com/wp-content/uploads/2012/04/tmp4B108.jpg

• Roth D. 2014. V(d)j recombination: mechanism, errors, and fidelity. microbiolspec 2(6): doi:10.1128/microbiolspec.MDNA3-0041-2014

• 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-phpapp02/95/neoplasia-robbins-path-33-728.jpg?cb=1341393120

References



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