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Advanced Genetics > V(D)J Recombination > Flashcards

V(D)J Recombination Flashcards

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1
Q

What is the purpose of V(D)J in its simplist form?

A
  • A process to generate antigen receptor diversity
  • One of the few processes where the cell deliberately permanently alters DNA - in antigen receptor (B and T cells)
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2
Q

How do CD4+ T cells and CD8+ T cells differ in their response?

A

CD4 + T cells:
- CD4+ T helper cell binds MHC 2-antigen complex on APC (antigen presenting cell) - both APC and T cell release cytokines + T cell clones itself
- Clones T cells produce diff cytokines that activate B cells and CD8+ cells

CD8+ T cells:
- Cytotoxic T cell interacts with MHC-1 epitope complex on infected cell - it produces granzymes & perforins
- Perforins form pores in plasma membrane
- Granzymes enter cell and break down proteins - lysing the cell

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3
Q

Outline of the structure on an antibody

A

C region - heavy chain (constant & variable regions)
V region - light chain + antigen binding region (constant & variable regions)
- Have no diversity (D) region

  • Hypervariable regions on both chains - generate high level of antigen specificity
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4
Q

How is the variable region generated?

A
  • By joining individual gene segments in the V(D)J recombination reaction
  • Any heavy chain can recombine with any light chain - required altering of antigen-binding pocket
  • This generates new antibody genes - antigen diversity
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5
Q

What is the function of the RAG proteins?

A
  • RAG1 & RAG2 - proteins that recognise recombination signal sequence (RSSs)
  • Need 2 molecules of RAG1/RAG2 - to bind RSS
  • RAG1 - active region in C-terminal core (active site DDE motif)
  • RAG2 - active region in N-terminus core (allosterically) - C-terminus has PHD finger that is critical for chromatin binding
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6
Q

Process of V(D)J recombination

A
  1. RAGs bind the RSSs and bring segments to be recombined at synaptic complex
  2. RAG1 causes nick on one strand; the resulting 3’OH attacks the opposite strand - direct transesterfication reaction
  3. Creates hairpin structrue at coding ends; a blunt DSB at the signal ends
  4. Coding ends and signal ends are processed differently
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7
Q

How are coding joints processed?

A

Coding Joints: - Hypervariable region 3

  1. Ku70:Ku80 binds DNA ends
  2. DNA-Pk: Artemis asymmetrically opens hairpin (SS nick) - generating palindromic P-nucleotides (reverse in complement nucleotides)
  3. N-Nucleotides are added by TdT onto ends of DNA (random - increasing diversity) ~20 nucleotides
  4. Strands are paired and unpaired nucleotides are removed by exonuclease
  5. Gaps are filled by DNA synthesis; ligation - to form coding join - adds to hypervariable region 3
  • Use NHEJ - but due to error prone nature - coding regions often loose or gain nucleotides
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8
Q

How are signal joints processed?

A

Signal Joints:

  1. Ku70:Ku80 binds DNA ends
  2. DNA ligase IV: XRCC4 ligates DNA ends = precise signal joint
  • Use non-homologous end joining (NHEJ)
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9
Q

When is TdT only expressed?

A
  • In pro B-cells and early T cells
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10
Q

What are the different stages where diversity can be generated?

A
  • Combinatorial joining of gene segments - V(D)J
  • Junctional diversification during gene segment joining - N and P nucleotides
  • Combinatorial joining of L and H chains
  • Somatic hypermutation + class switch recombination
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11
Q

What chain are N nucleotides added more to?

A

Heavy chain

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12
Q

How are heavy chains IgM and IgD expressed by naive B cells?

A
  • Heavy chains IgM and IgD are expressed by naive B cells via alternative splicing
  • Alternatice splicing - is RNA - moves introns in constant region
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13
Q

How is antigen receptor diversity obtained in a pro-B cell, and what stages does it go through to become a mature naive B cell?

A
  • Pro-B cell - first - productive rearrangement of heavy chain - increases heavy-light chain combinations you can make = diversity (V(D)J)
  • Pre-B cell
  • Immature naive B cell
  • Mature naive B cell - circulation through lymph organs
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14
Q

How do the different immunoglobulins differ?

A
  • They differ in their interactions with complement and Fc receptors
  • They differ in their locations and ability to other cells in immune system
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15
Q

How can you switch to IgG, IgE or IgA?

A
  • Requires deletion of intervening DNA via class switch recombination
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16
Q

Briefly explain the primary and secondary responses of B cells and where Class Switch Recombination (CSR) and Somatic Hypermutation (SHM) occur?

A
  • Stem cells in bone marrow (Ig V(D)J gene rearrangement)
  • Stimulation of B cells (with antigens) via T cell signalling

Primary response - maturation process (in germinal centres of lymph nodes) - clonal expansion - CSR & SHM
- CSR - swap heavy chain constant region - doesnt alter affinity of antibody or antigen
- SHM - Iterative process - more and more mutations until highest affinity binding
- Plasma cells - secretes antibodies

Secondary Response - clonal expansion & more CSR & SHM

17
Q

Class Switch Recombination (CSR) - Process

A
  1. Transcription through switch region initiated - by promoter upstream
  2. AID modifies DNA in switch region - allowing class switching (AID makes G-U mismatches - deamination)
  3. UNG (removes uracil - abasic) and APE1 (excises ribose - ss nicks) recognise G-U mismatches introduce cluster SS nicks on both DNA strands
  4. DNA-Pk and other repair proteins act to intiate DSBR
  5. DSBR machinery joins the two switch regions - and excises intervening sequences
  6. The selectred constant region is now located adjacent to the V(D)J region - crossing over
18
Q

Where does somatic hypermutation predominantly generate antigen diversity?

A

In the hypervariable regions

19
Q

How do SHM and CSR compare?

A

CSR:
- Requires DSBs
- High AID-density
- Excise large block of DNA

SHM:
- SSB is probably sufficient for mutation
- Low depedence of AID-density fusions
- Point mutation - excise small amount of DNA

20
Q

What 3 different ways can SHM occur once AID has nicked?

A

SHM occurs in mature B cells:
1. Replication over G-U mismatch (causes G-A transition)
2. Uracil DNA-glycosylase - creates abasic site - error prone polymerase inserts random bases opposite abasic site
3. Excise small block of DNA around G-U mismatch - recuritment of MMR elements leads to insertion of mutations - preferentially at A:T

21
Q

How is AID used in SHM?

A
  • Used only to get mutation in variable exon - not constant region (directed towards variable exon via transcription factors)
  • AID deaminates cytidine to uridine (without sugars attached)
22
Q

Why do you get more DSBs in switch regions?

A
  • G rich switch regions create R loops - so takes longer
  • Therefore more likely to get AID activity - and get DSBs
23
Q

What is the key similarities/differences between B and T cell receptors?

A

Similarities:
- T cell receptors have binding site similar to antibody
- Both use V(D)J recombination - T cells use (12/23 rule)
- Same RAG proteins involved

Differences:
- T cell receptors are ALWAYS membrane-bound
- T cell receptors have a single antigen binding site
- T cell receptors recognise antigen in conjunction with MHC molecules
- T cell receptors do not undergo SHM or CSR
- In T cell receptors - much more diversity lies in hypervariable region 3 - and is generated via elevated junctional diversity and the larger number of J gene segments

24
Q

How is RAG cutting regulated?

A

RAG1/2 recognise the same RSS’s in both B and T cells
- RSS’s are only made accessible to RAGs in correct cell type
- RSS’s are only made accessible to RAGs at correct stage of lymphocyte development
- RSS’s are only made accessible to one of the two alleles

25
Q

How is V(D)J restricted to G1?

A
  • By phosphorylation-degradation of RAG2
  • RAG1 is active through whole time
  • But RAG2 is suppressed throughout S, G2 and M - then activated in G1
26
Q

Why do you need to alter the nucleosome to initiate V(D)J?

A
  • Becasue RAG1/RAG2 can’t cut when RSS is contstrained within a nucleosome
  • Nucleosome inhibits cutting - DNA polymerase is too big to pass through nucleosome
27
Q

Where is H3K4me3 often found?

A

H3K4me3 is an active chromatin mark often found at promoters
H3 = Histone 3
K4 = lysine - position 4
me3 = trimethylated

28
Q

How is RAG2 recruited to the nucleosome?

A

PHD domain on RAG2 interacts with tri-methylated H3K4 (H3K4me3)
- But still isn’t enough to remodel nucleosome, allow RAGs to cut and initiate recombination

histone acetylation alone is not enough either

29
Q

How is recombination initiated in the nucleosome?

A
  • Non-coding transcripts traverse RSSs of all antigen receptor loci; insertion of transcription terminator prevents non-coding transcription
  1. Transcription is essential for recombination - triggering:
    - Increased histone acetylation - increasing chromatin acessibility
    - Increase H3K4me3 = recruitment of RAGs via RAG2 PHD domain
    - But these processes (alone/together) are not enough to trigger recombination
  2. As DNA polymerase approaches nucleosome
    - H2A/2B dimer is removed - making ~40 bases of DNA accessible - creating a hexasome - short window where RAGs can bind
    - If RSS happens to be in this small region that is made acessible - RAGs will cut
    - This makes RSSs acessible transiently - helps protect DNA from too many cuts from RAGs
30
Q

Why are recombination enhancers important in regulating V(D)J?

A
  • They are only active in correct cell lineage
  • Are activated at correct stage of development
  • Can contact the promoters to activate transcription - promoters
  • Can explain lineage/stage specificity of activation
31
Q

How can we explain allelic exclusion? (The restriction of expression to one allele per cell)

A

Allelic exclusion - only get recombination of one chromosome - e.g., if first chromosome is not sucessfull then next one is activated
-Inactive allele associates with pericentric heterochromatin
- Active allele goes to active region of nucleus (centre)
- If rearrangement is non-productive - then other allele can be activated

32
Q

What is cryptic recombination, and how can it lead to cancer?

A
  • Recombination with a cryptic RSS outisde of the antigen receptor loci
    BUT:
  • If cryptic recombination (RAGs cut) occurs near an oncogene - can cause overexpresison of this oncogene
  • Is a prominent way to get chromosome translocation/recombination - e.g., LMO2, SIL/SCL, TCR/IgH inversion
33
Q

What is end donation, and how can it lead to cancer?

A
  • When a broken end on other chromosome becomes aberrantly recombined into antigen receptor loci
  • 50% for RSS on both sides, 50% for RSS on just one side
  • E.g., BCL-2/IgH - most common translocation in human cancer - prevents cell apoptosis
34
Q

How can the excised bit of DNA from V(D)J be dangerous?

A

Transposition/Re-integration
- Is very similar to transposition reaction - same transesterfication reaction - except the target site is an RSS
- RAGs cut out bits of DNA which can be placed randomly via staggered insertion - generating a duplication at the insertion point
- Happens often at cryptic RSSs - but doesnt cause cancer as it will often be in silent parts of the genome

35
Q

What 4 ways can V(D)J errors cause cancer?

A
  • Cryptic recombination
  • End donation
  • Transposition
  • Re-integration
36
Q

How can CSR errors cause cancer?

A
  • Translocation errors in CSR
  • Errors of SHM
37
Q

How can errors in CSR lead to oncogene activation?

A
  • Translocations of C-myc gene to IgH switch region appears to be error in CSR (AID-dependent)
  • Results in aberrant MYC activation - and Burkitt’s Lymphoma
38
Q

How can SHM lead to oncogene activation?

A

Mis-targeting of AID - onto oncogenes in B cells
- Leads to oncogene activation
- E.g., tumour suppressor genes ARF, INK4B / Oncogenes: MYC, BCL6

Advanced Genetics (4 decks)

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