A Prototype Ig Gene: Murine Kappa IMMUNOGLOBULIN GENES UNDERGO TWO DNA About 10 0 4 J Gene 1 C κ Gene Segment s Vκ gene segments Segmen t REARRANGEMENTS V(D)J Recombination: Generates Diversity both light and heavy chains Multiple V gene segments, distant from J and C A few J gene segments One C gene segment

“GERMLINE” Ig genes are NOT transcribed or translated. Changes Class switch recombination: Isotype (antigen elimination) heavy chains only

V(D)J Recombination in the Kappa Locus

About 10 0 4 J Gene 1 C κ Gene Segment s Vκ gene segments Segmen t

DNA is deleted

V J C

DNA Rearrangement RemovesSequences Between V, D and J Segments V(D)J recombination RNA Splicing Removes SequencesFigure Between 4-2 J and C Segments Figure 4-6 involves DELETION of DNA between V, D and J coding DNA segments.

The deleted DNA is RNA LOST from the cell because it is not replicated.

1 Recombination Signal Sequences (RSSs) Flank RearrangingFigure Gene 4-5 Segments

RSS=heptamer, spacer 7bp 12/23 bp 9bp and nonamer

RAG Proteins: Lymphocyte Specific and Uniquely Required for VDJ Recombination

RAG (Recombinase activating gene) 1 and 2 proteins INITIATE VDJ recombination:

1. Bind to the RSS sequences

2. Stabilize the synapse between two segments

3. Introduce a nick between coding region and RSS sequence; subsequent trans-esterification leads to hairpin structures on the coding sequences and blunt ends on the RSS sequences.

Components of general DNA repair FINISH VDJ recombination:

Ku70/80

DNA-dependent protein kinase

Artemis

XRCC4

DNA ligase IV

2 Addition of Bases at V-D-J Junctions: #1: #2: An Important Source of Diversity Lymphocyte- Figure 4-7 Constitutive Figure 4-8 “N” Specific addition

Initiation of Resolution V(D)J of cleavage rearrangement : products: RAG- DNA non- dependent homologous “P” cleavage end-joining addition machinery

Figure 7-18

B Menu F

CONSEQUENCES OF V(D)J RECOMBINATION 4. Activates transcription of the rearranged gene Juxtaposition of intronic enhancers with V region 1. Combinatorial diversity: # of possible combinations promoters. is the product of the # of recombining segments i.e. for mouse h.c.: 120x20x4=104 E 2. Junctional diversity at CDR3 Unrearranged V gene segments J’s ENHANCER C Deletion of bases at junctions Each has an inactive promoter N region additions at junctions VJ recombination P region additions at junctions

3. Allows receptor editing to alter potentially self-reactive E antibodies

Enhancer activates the promoter of the rearranged V gene, resulting in transcription.

3 Figure 4-15 part 1 of 2 Figure 4-18

Figure 4-17 Ig HEAVY CHAIN LOCUS

1. Membrane vs secreted exons RNA

2. Mu and delta isotypes

3. Gamma, epsilon and alpha isotypes CLASS SWITCH RECOMBINATION (CSR) A second DNA rearrangement, unique to the HC locus

IgM and IgD Are Generated from a Single Primary Transcript by DIFFERENTIAL mRNA POLY A/SPLICING mRNA FigureDNA 4-19 rearrangement: CSR Figure 4-20 Splicing

4 Membrane vs. Secreted Mu CSR Involves DNA Deletion and Loss mRNAs encoding both Figure 4-21 membrane and secreted forms of mu heavy chain are gen- erated from a single primary transcript by differential splicing and polyadenylation

“Germline” (I region) Transcripts Are Necessary Cytokines regulate I region transcription: For Isotype Switch Recombination Figure 9-7

T cell secretes cytokines TGFβ

AID (Activation Induced Deaminase) is required

Specific I region transcription IαSαCα RNA for CSR

Deaminate ssDNA displaced by I transcript? Isotype switch recombination to Cut and join specific C gene segment Initiates cleavage? H Sµ and Sα DNA

Subsequently, some repair proteins are involved: VDJCα mRNA Ku70/80, XRCCR

IgA

5 V(D)J Recombination CSR 1. Humans with mutations in gene products required for Join in exon Join in intron V(D)J recombination are immunodeficient:

RAGs required RAGs Not required RAG Various SCIDs, including Omenn’s AID is required syndrome

Repair enzymes Repair enzymes Artemis Radio-sensitive SCID

Generates diversity Changes isotype Ligase IV SCID with developmental deficiency Ag specificity Ag elimination

2. Humans with mutations affecting CSR have hyper-IgM Random Regulated by T cell AID mutations and other mutations signals

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