Antigen Receptor Gene Assembly Flashcards
The Paradox
How can precursor lymphocytes generate a library of Ig and TCR with >10^9 distinct variable regions?
- Dogma held that each 1 gene = 1 protein.
- Need >1/3 of our genome for Ig genes alone.
What is the main objective of the somatic V(D)J recombination?
- To generate a vast diversity of antigen receptors (antibodies in B-cells and T-cell receptors in T-cells) that can recognize and bind to an enormous variety of pathogens and foreign molecules.
V(D)J Recombination
Variable Diversity Joint
Mechanism of somatic recombination that occurs only in developing lymphocyte during the early stages of T and B cell maturation
The challenge of adaptive immunity
Variable pathogens need recognizing, all with different antigen epitopes
B and T Cell Receptor
DNA rearrangements that generate functional immunoglobulin (Ig) and T cell receptor proteins are indispensable for the development, progression, and survival of B and T cell precursos.
Immunoglobulins
(Ig)
- Are specifically associated with B-cells
- Produce by B-lymphocytes and secreted into the plasma.
- The Ig molecule in monomeric form is a Y-shaped glycoprotein.
- Disulfide bonds
T-cell Receptor
(TCR)
- Membrane bound heterodimeric proteins normally consisting of the highly variable alpha and beta chain.
- The minority of T-cell express an alternate receptor, formed by variable gamma and delta chains.
T-Cell Receptor
(TCR)
- TCR Beta and Delta chains are simialry encoded by distincits sets of V(D)J.
- TCR Alpha and Gamma chains are encoded by sets of V and J genes.
AgR Gene Assembly by VJ recombination
Light Chain
- Somatic Recombination in germline DNA to rearrange V-J
- Transcription
- Primary transcript RNA gets spliced to remove introns
- mRNA-combined VJC regions
- Translation leads to polypeptide chain
AgR Gene Assembly by V(D)J recombination
Heavy Chain
- Somatic recombination in germline DNA that rearrange D/J and joins them.
- Somatic recombination of V-DJ joined rearranged DNA.
- Transcription to primary RNA.
- Splicing to mRNA combines VDJC.
- Translation to polypeptide chain.
How does recombination take place?
Recombination Signal Sequences
(RSSs)
- Generates the diversity of antigen receptors in B-cells and T-cells.
- These sequences guide the enzymes involved in recombination, particulary the RAG1 and RAG2 proteins, to the correct locations in the DNA to cut and join the V(D)J gene segments.
- During V(D)J recombination , double-stranded breaks are generated between the heptamer of the RSS and the adjacent V, D, or J coding sequence.
- 12-23 rule-restricted to recombine with each other in light chain.
- Their relative orientation determines whenether the reaction proceeds by inversion or deletion.
The V(D)J Recombination
Cleavage Phase
(RAG1/RAG2)
- Two compatible gene segments (RSSs) V&J
- Form synapsis and they are bound by recombinases R1/R2 (RAG)
- Binding activates endonuclease activity and cleaves separating DNA, making V&J hairpins ends (G.O.D).
G.O.D complex binds RSS, synapsis of complexes are made , and then it cleaves on RSSs
The V(D)J Recombination
Joining Phase
(NHEJ pathway)
DNA repair complexes join ends:
* Signal joint containing RSSs and forms extra chromosomal circles, eventually degrade and are lost from genomes
* Coding joint 1/3 in frame.
Types of Reaction
(RSSs)
- Deletion - RSS are in same orientation (23 & 12), gets deleted at signal joint, coding joint remains.
- Inversion - RSSs in opposite strans, begins direction of transcription.
Tools available to study VDJ recombination
Episomal Substrates:
Deletion
Two heptamer/nonamer Ig joining signal sequences flank prokaryotic transcription terminator.
Signals flanked by E.Coli plac promoter and cat gene (resistance)
Terminator: Prevents expression of resistance gene, no recombination = no drug resistance/die in E.Coli. (After VDJ recomb in lymphoid cells, resistance gene deleted.
1. Substrate DNA transfected into eukaryotic cells, includes lymphoid progenitors.
2. Recovered 48hrs after
3. Introduced into E.Coli by transformation. Ratio of doubley resistant (CAMr/AMPr) = fraction of DNA rearranged at joining signals.
Recombinase activity is restricted to pre-B/T-cell lines
Tools available to study VDJ recombination
Stable Substrates:
Inversion
Genomic DNA + marker plasmid transfected into DGR fibroblast line.
GPT is antisense relative to promotor
After recombination, inversion of GPT allows for correct orientation.
NHEJ Factors:
Repair of RAG break
DNA-dependent kinase catalytic subunit (DNA-PKcs complex: Ku70, Ku86, and DNA-PKcs.
* Ku70/Ku86 have DNA end binding activity.
* Required for signal and coding joint formation.
* DNA-PKcs is a serine/threonine kinase required for coding, but not signal, joint formation.
Artemis.
* Opens hairpin-sealed coding end (DNAPKcs-dependent)
DNA Ligase IV, XLF, and XRCC4
* Required for signal and coding joint formation
Severe block in B/T cell development in mice deficient for any of these NHEJ factors.
Generation of Diversity
Combinatorial Diversity
TCR
- αβ TCR repetoire ~10^9
- 5k distinct TCRα variable regions from 100 Vα joining 50 Jα
- 540 distinct TCRβ variable regions from 2Dβ , 9Jβ and 30 Vβ
- 2x10^6 αβ TCR combinations.
- The number of potential combinations for the heavy chain alone is: 40V x 25D x 6J= 6k different combinations.
Generation of Diversity
Junctional Diversity
- Refers to the additional genetic variation that is introduced at the joining regions between the V (Variable), D (Diversity), and J (Joining) gene segments during somatic V(D)J recombination.
- Generates immense variability in the hypervariable regions (CDR3) of both B-Cell and T-Cell receptors, which are the regions that directly interact with antigens.
- 1/3 of junctions are in reading frame (w/ATG), 2/3 nonfucntional
Generation of Diversity
Junctional Diversity
Summary
- DNA cleavage by RAG1 and RAG2
- Open hairpin by artemis /DNApk and creates Palindromic (P) element (a cut back into the sequence=overhang).
- Addition of nucleotides by TdT. Terminal dideoxytranferase –> job = adds random nucleotides to knicked hairpins –> non-templated regions (N).
- Joining by polymerase, DNA ligase IV, XLF, XRCC4
Evolution of the adaptive immune response
- Seven different receptor loci: IgLkappa, IgLlamdba, IgH, TCRalpha, TCRbeta, TCRgamma, TCRdelta.
- Was previously though that RAG was a transposbale element in prokaryotes, captured by proteins/arrays throught evolution.
Order TCR Gene Assembly
D-J Beta and V-DJ Beta rearrangment occurs at DN T-cell stage in thymus.
* TCRb = developmental checkpoint be functional for DP.
* If this rearrengment is functional, pre-TCR drives proliferation.
V-J alpha rearrangment occurs during DP stage
* If successful, it shuts off TCR rearrengment and becomes SP.
Order Ig Gene Assembly
D-J(H) occurs in early pro-B cell
V(H) - DJ(H) occurs in late pro-B cell
* Success = pre-BCR, proliferation
V(k) - J(k) and V(lambda) - J(lambda) occurs in small pre-B cell, if success - immature B-cell, IgM in Bone Marrow.
Accessibility
Problem: All V gene loci present common substate pairs and common recombinase (RAG-1/2) at all stages of pre-B and pre-T cell development
The solution are the recombination events that are controlled by modulating the availability of loci and regions within a locus to the common recombinase (chromatin accessibility model).
Refers to the regulation of chromatin structure and gene expression that controls whether specific gene segments (V, D, or J) are available for recombination.
This process ensures that the recombination machinery (RAG-1/2) can access and rearrange gene segments only at appropiate times and in specific cell types.
Modulation of Accessibility
Regulated by cis - acting elements
Correlates with transcription as well as the chromatinf landscape of gene segments
* Histone modifications: methylation and acetylation
* DNA methylation (CpG)
Chromatin Nods
RAG association with Chromatin
- RAG1 binds directly to RSSs, while RAG2 enhances this process and links the recombination machinery to transcrriptionally active, open chromatin marked by histone modifications like H3K1me3.
- RAG2’s recognition of H3K4me3 is key to targeting active gene loci for recombination.
- Chromatin accessibility is regulated by histone modifications, chromatin remodeling complexes, and transcriptors factors, which together ensure that the V(D)J gene segments are accesible at the correct development stages.
Mechanism Allelic Exclusion
Enforced at IgH, IgL, TCRbeta loci by feedback inhbition from AgR signals.
In nucleus rearrangement:
* If first allele is nofunctional –> rearrange second
* If functional: displayed on surface, and sends feedback signals to nucleus to shut down rearrange of TCRbeta. Drives proliferation.
Enforced in DN, cannot move to DP if no feedback occurs.
TCR beta unaccessible after TCRalpha rearrangement in DO thymocytes.
TCR beta EaKI =/ rearrangement
Maintaing Genome Stability
NHEJ
Leads to normal number of diverse lymphocytes.
* RAG cleavage = ~60x10^6/hr in BM aline
* Efficient recognizing of breaks
* Chromosomal translocations, large deletions can cause oncogene = lymphoid tumors.
Maintaing Genome Stability
Cell cycle checkpoint activation
Prevents cell cycle progression
Cause apoptosis if repair fails
ATM recognizes ds breaks, if breaks nor repaired it phosphorylates a lot and enforces checkpoints.
Chronic phosphorylation on Chk2/p53 triggers cell death
Checkpoint/DNA repari defects = cancer risk
